Recombinant DNA encoding a eukaryotic tyrosine kinase target protein

ABSTRACT

A novel expression cloning method is provided for the detection, identification and purification of target proteins capable of binding at least to a tryosine-phosphorylated domain of a eukaryotic tyrosine kinase using novel peptide probes comprising an amino acid sequence substantially corresponding to a portion of a tyrosine-phosphorylated domain of a tyrosine kinase. The probe has at least one phosphorlated tyrosine residue and may be detectably labeled. Also disclosed is a method for preparing the probe, a method for mapping to a chormosome a gene encoding a protein capable of binding to tyrosine-phosphorylated domains of tyrosine kinases, and a method for purifiying such a protein with the probe. Non-limiting examples of novel proteins discovered using the above cloning method include GRB-1, GRB-2, GRB-3, GRB-4 and GRB-7, as well as nucleic acid encoding these proteins, and methods for detecting these proteins are also provided.

This is a division of application Ser. No. 07/906,349, filed Jun. 30, 1992, now U.S. Pat. No. 5,434,064, which is a continuation of application Ser. No. 07/643,237, filed Jan. 18, 1991, now abandoned, the entire contents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention, in the field of molecular and cell biology, relates to a novel method, based on direct expression cloning, for identifying target proteins capable of binding to and/or serving as substrates for receptor or cytoplasmic tyrosine kinases. The invention also relates to novel proteins identified using this method.

2. Description of the Background Art

A variety of polypeptide growth factors and hormones mediate their cellular effects by interacting with cell surface receptors and soluble or cytoplasmic polypeptide containing molecules having tyrosine kinase enzymatic activity (for review, see Williams, L. T. et al., Science 243:1564-1570 (1989); Ullrich, A. et al., Cell 61:203-212 (1990); Carpenter, G. et al. J. Biol. Chem. 265: 7709-7712 (1990)). The interaction of these ligands with their receptors induces a series of events which include receptor dimerization and stimulation of protein tyrosine kinase activity. For the epidermal growth factor receptor (EGFR) as well as other receptors with tyrosine kinase activity, such as the platelet-derived growth factor receptor (PDGFR), kinase activation and receptor autophosphorylation result in the physical association of the receptor with several cytoplasmic substrates (Ullrich et al., supra).

Two substrates for the EGFR kinase have now been definitively identified in living cells: (a) the phosphatidylinositol specific phospholipase C-δ (PLC-δ) and (b) the GTPase activating protein (GAP), a protein which may be in the effector loop of the ras protein (Margolis, B. et al. Cell 57: 1101-1107 (1989b); Meisenhelder, J. et al. Cell 57: 1109-1122 (1989); Molloy, C. J. et al. Nature 342: 711-714 (1989); Wahl, M. I. et al. J. Biol. Chem. 265: 3944-3948 (1990); Ellis, C. et al. Nature 343: 377-381 (1990); Kaplan, D. R. et al. Cell 61 121-133 (1990)).

Similarly, activated PDGFR was shown to tyrosine phosphorylate, and to become associated with PLC-δ, GAP, and cellular tyrosine kinases such as pp60^(src) (Gould, K. L. et al., Molec. Cell. Biol. 8:3345-3356 (1988); Meisenhelder, J. et al., Cell 57:1109-1122 (1989); Molloy, C. J. et al., Nature 342:711-714 (1989); Kaplan, D. R. et al., Cell 61:121-133 (1990); Kazlauskas, A. et al., Science 247:1578-1581 (1990); Krypta, R. M. et al., Cell 62:481-492 (1990); Margolis, B. et al., Science 248:607-610 (1990). While the exact sites responsible for the association of EGFR with either PLC-δ or GAP have not been completely clarified, recent work has begun to identify regions on both the substrate and receptor which contribute to the association.

SH2 (src homology 2) domains appear to be the regions responsible for the association of several tyrosine kinase substrates with activated growth factor receptors. SH2 domains are conserved sequences of about 100 amino acids found in cytoplasmic non-receptor tyrosine kinases such as pp60src, PLC-δ, GAP and v-crk (Mayer, B. J. et al., Nature 332:272-275 (1988); Pawson, T. Oncogene 3:491-495 (1988)). While having distinct catalytic domains, all these molecules share conserved SH2 and SH3 (src homology 3) domains and the ability to associate with receptors with tyrosine kinase activity (Anderson, D. et al., Science 250:979-982 (1990)).

Tyrosine kinase activation and receptor autophosphorylation are prerequisites for the association between growth factor receptors and SH2 domain-containing proteins (Margolis, B. et al., Mol. Cell. Biol. 10:435-441 (1990); Kumjian et al., Proc. Natl. Acad. Sci. USA 86:8232-8239 (1989); Kazlauskas, A. et al., Science 247:1578-1581 (1990)). In particular, the carboxy-terminal (C-terminal) fragment of the EGFR, which contains all the known autophosphorylation sites, binds specifically to the SH2 domains of GAP and PLC-δ (see below). Hence, a major site of association exists between the SH2 domain of these substrate proteins and the tyrosine phosphorylated C-terminal tail of the EGFR.

With the recognition that binding to the activated tyrosine kinase receptor is conserved among several substrate proteins, efforts to identify additional substrates which share these properties have been undertaken. Target proteins which bind to activated receptors have been identified by analysis of proteins that co-immunoprecipitate with growth factor receptors, or that bind to receptors attached to immobilized matrices (Morrison, D. K. et al., Cell 58:649-657 (1989); Kazlauskas, A. et al., EMBO J. 9:3279-3286 (1990)). While the identity of some of these proteins is known, several others detected utilizing these approaches have not been fully characterized. Moreover, it is possible that rare target molecules which interact with activated receptors have not been detected due to the limited sensitivity of these techniques; the actual stoichiometry of binding may be low, and the detergent solution necessary to solubilize proteins may disrupt binding.

Conventional approaches to isolate and clone these proteins have been arduous, requiring the use of large quantities of tissue or cells lines to purify sufficient amounts of protein for microsequence analysis and subsequent conventional cDNA cloning. Therefore, a need for new approaches for the cloning and subsequent isolation and identification of these proteins is recognized in the art.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the deficiencies of the related art.

It is also an object of the present invention to understand and gain control over the regulation of cell growth and oncogenesis by providing the ability to identify target proteins for tyrosine kinases, including both receptor and cytoplasmic tyrosine kinases in eukaryotic organisms.

It is a further object of the present invention to provide a novel expression/cloning system for the rapid cloning of target proteins which bind tyrosine kinase proteins which are present intracellularly and in cell receptors of eukaryotes. The cloning method is based on the ability of a certain class of substrates to bind specifically to the tyrosine-phosphorylated carboxy-terminus (C-terminus) of the proteins having tyrosine kinase activity. Non-limiting examples include proteins that bind at least one of cytoplasmic and receptor tyrosine kinases, such as a receptor tyrosine kinase found in epidermal growth factor receptor (EGFR) (see Example VI, below).

Another object of the present invention is to provide a method of cloning tyrosine kinase target proteins, which method important advantages over conventional cloning methods, including avoidance of the laborious and costly task of purifying potential target proteins for microsequencing analysis.

Another object of the present invention is to provide a method for identifying receptor target molecules having tyrosine kinase activity whose association with activation receptors could not otherwise be detected using conventional techniques.

Another object of the present invention is to provide for the identification of structurally or functionally related proteins which, though only weakly homologous at the nucleic acid level, are similar in their property of binding to activated receptors with tyrosine kinase activity, which latter ability is important since conventional screening methods used to identify related genes are typically based on low stringency nucleic acid hybridization. Conventional hybridization-based screening would not have been successful in cloning and identifying such tyrosine kinase target proteins of the present invention, exemplified as non limiting examples as GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7, because of their lack of similarity at the DNA level.

The methods of the present invention take advantage of the discovery that the C-terminus of the EGFR protein in which the tyrosine residues are phosphorylated can bind substrates as described herein. By creating a labelled polypeptide which substantially corresponds to at least a portion of phosphorylation domain of a tyrosine kinase, a probe is provided having at least one phosphorylated tyrosine. Such a probe can be used to detect, identify and/or purify target proteins from solutions or as part of screening of cDNA expression libraries from eukaryotic cells or tissues. Such tyrosine kinase target proteins, discovered according to the present invention, are termed "GRB" (for Growth factor Receptor Bound) for the initial receptor tyrosine kinases used, but which target proteins are not limited to growth factor receptors. Accordingly, GRBs of the present invention include target proteins for any eukaryotic tyrosine kinase which are provided according to the present invention.

The novel cloning methodology of the present invention has been designated, "CORT" (for Cloning Of Receptor Targets), and may also be applied to detecting, identifying, cloning or purifying target proteins for any tyrosine kinase, such as a soluble, cytoplasmic or receptor tyrosine kinase.

The method of the present invention is proposed as a novel approach having both generality and rapidity for the identification and cloning of target molecules for tyrosine kinases.

The present invention is thus directed to a method for detecting a target protein in solution, which is a target of a receptor or cytoplasmic tyrosine kinase, the target protein being capable of binding to at least a portion of a tyrosine-phosphorylated polypeptide of the receptor or cytoplasmic tyrosine kinase, the method comprising:

(a) contacting the solution (as a cell, an extract thereof, a lysate thereof, or a supernatant thereof) with a solid phase carrier, causing the binding of the protein to the carrier to provide a carrier-bound target protein;

(b) incubating the carrier-bound target protein with the tyrosine-phosphorylated polypeptide, which has been detectably labeled, allowing the polypeptide to bind to the carrier-bound protein;

(c) removing materials not bound to the carrier-bound target protein;

(d) detecting the presence or measuring the amount of the tyrosine-phosphorylated polypeptide bound to the carrier,

thereby quantitatively or qualitatively detecting the target protein in said solution.

In one embodiment, the receptor or cytoplasmic tyrosine kinase is any eukaryotic tyrosine kinase, such as epidermal growth factor receptor, the platelet-derived growth factor receptor, or the fibroblast growth factor receptor pp60^(v-src), pp160^(gag-abl), pp130^(gag-fps), pp59^(c-fyn), PDGF receptor B, CSF-1 receptor, pp150^(c-fms), pp150^(v-fms), EGF receptor, Insulin Receptor, IGF-1 receptor, pp68^(gag-ros), PLC-δ, middle t-pp60^(c-src) middle t-pp62^(c-yes), and the consensus sequences EEEEEY(PO₄)MPMXX (SEQ ID NO:11), EEEEEY (PO₄)VPMXX (SEQ ID NO:12), DDDDDY(PO₄)MPMXX (SEQ ID NO:13), and DDDDDY(PO₄)VPMXX (SEQ ID NO:14) or a phosphorylatable fragment thereof, preferably a polypeptide of about 10 to 250 amino acid residues, more preferably 10 to 40 or 15 to 50 residues, wherein the polypeptide is produced recombinantly, synthetically or by enzymatic digestion of a purified tyrosine kinase molecule.

This method is preferably performed using a prokaryotic cell, most preferably a bacterial cell such as E. coli. The cell may also be eukaryotic, such as a yeast or a mammalian cell.

Preferably, the phosphorylated polypeptide is detectably labeled.

The solid phase carrier can be any material which can be used to bind a target protein for a tyrosine kinase. The carrier may preferably be a nitrocellulose membrane, such as to which are transferred proteins released for lysed bacterial cells when a library is being screened.

The present invention also provides a method for mapping to a eukaryotic, such a mammalian, human, murine, or other eukaryotic chromosome a gene encoding a protein which is capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase molecule, the method comprising:

(a) infecting a host or host cells which a eukaryotic gene expression library;

(b) detecting a clone expressing the protein using a method according to claim 1;

(c) sequencing the DNA of the clone; and

(d) mapping the sequence to a eukaryotic chromosome.

The present invention is also directed to a polypeptide probe useful in the detection of the expression of a protein capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase. The probe comprises an amino acid sequence derived from the tyrosine-phosphorylated portion of the receptor or cytoplasmic molecule, or a functional derivative thereof, lacks the tyrosine kinase domain, and the sequence must contain at least one phosphotyrosine residue, such as 1,2,3,4,5,6,7,8,9,10 or 11 phosphotyrosines. The probe should be detectably labeled with known labels.

A preferred probe has between about 10 and 250 amino acid residues, preferably 10-35, 16-30, 21-35, 15-35, or 20-40 residues.

The probe of the present invention is useful for detecting target proteins for receptor or cytoplasmic tyrosine kinases including but not limited to, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), colony stimulating factor-1, (CSF-1), insulin receptor, phospholipase C-δ (PLC-δ) and insulin like growth factor-1, (IGF-1), pp60^(v-src), pp160^(gag-abl), pp130^(gag-fps), pp59^(c-fyn), PDGF receptor B, CSF-1 receptor, pp150^(c-fms), pp150^(v-fms), EGF receptor, insulin receptor, IGF-1 receptor, pp68^(gag-ros), PLC, middle t-pp60^(c-src), middle t-62^(c-yes), and the consensus sequence EEEEEY(PO4)MPMXX (SEQ ID NO:11), EEEEY (PO4)VPMXX (SEQ ID NO:12), DDDDY (PO4)MPMMXX (SEQ ID NO:13), and DDDDDY(PO4) VPMXX (SEQ ID NO:14) or a phosphorylatable fragment thereof, e.g., as described Cantley et al., Cell 64:281-302 (1991) or Ulrich and Schlessinger Cell 61:203-312 (1990), which references are entirely herein incorporated by reference.

The present invention also includes a method for preparing the above probe, comprising

(a) providing the receptor or cytoplasmic tyrosine kinase, or a recombinantly, enzymatically or synthetically produced fragment thereof, wherein the receptor or cytoplasmic tyrosine kinase, or fragment thereof, has both a tyrosine kinase domain and a tyrosine-phosphorylated domain, the tyrosine-phosphorylated domain including at least one tyrosine residue capable of being phosphorylated by the tyrosine kinase;

(b) incubating the receptor or cytoplasmic tyrosine kinase, or fragment, with detectably labeled adenosine triphosphate under conditions permitting phosphorylation of the tyrosine residue, causing phosphorylation of the tyrosine residue thereby producing the probe. In a preferred embodiment, the method includes the step of:

(c) additionally treating the phosphorylated receptor or cytoplasmic tyrosine kinase molecule with an agent capable of cleaving the molecule between the tyrosine kinase domain and the tyrosine-phosphorylated domain.

A preferred cleaving agent is cyanogen bromide.

In another embodiment, the above method involves a genetically engineered receptor-like derivative which is a polypeptide encoded by a DNA molecule comprising a DNA sequence encoding tyrosine kinase, linked to a DNA sequence encoding a selective enzymatic cleavage site, linked to a DNA sequence encoding the tyrosine-phosphorylated domain, and wherein the agent is an enzyme capable of cleaving at this cleavage site. Preferred enzymes are Factor Xa and thrombin.

Also provided is a method for purifying from a complex mixture a protein which is capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase molecule, the method comprising:

(a) contacting the complex mixture with a solid phase carrier to which a probe is bound, allowing the protein to bind to the probe;

(b) removing materials not bound to the carrier; and

(c) eluting the bound protein from the carrier,

thereby purifying the protein.

The present invention is also directed to a protein, GRB-1, having the amino acid sequence shown in FIG. 4 (SEQ ID NO:2). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-2, which includes the amino acid sequence shown in FIGS. 26A-26C ((SEQ ID NO:6). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-3, which includes the amino acid sequence shown in FIGS. 34A-34C (SEQ ID NO:4). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-4, which includes the amino acid sequence shown in FIGS. 35A-35B (SEQ ID NO:8). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-7, which includes the amino acid sequence shown in FIGS. 36A-36G (SEQ ID NO:10).

The invention is also directed to a DNA molecule encoding a polypeptide having a amino acid sequence substantially corresponding to the amino acid sequence of at least one of GRB1, GRB-2, GRB-3, GRB-4 or GRB-7 proteins. Included are DNA molecules encoding functional derivatives of these proteins. When the DNA molecule naturally occurs, it is substantially free of the nucleotide sequences with which it is natively associated. The DNA molecules of this invention may be expression vehicles, such as plasmids.

Also provided is a host transformed with each of the above DNA molecules.

The present invention also includes a process for preparing a target protein substantially corresponding to the amino acid sequence GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 protein, comprising:

(a) culturing a host comprising a recombinant nucleic acid having a nucleotide sequence encoding the target protein under culturing conditions such that the target protein is expressed in recoverable amounts; and

(b) recovering the protein from the culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a filter blot pattern showing that the carboxy-terminus of the EGFR interacts with GAP-SH2 immobilized on nitrocellulose filters, Bacterially-expressed trpE/GAP-SH2 fusion protein or trpE as a control was spotted at various concentrations onto nitrocellulose filters. The filters were hybridized overnight with (³² P)-labelled C-terminal domain of the EGFR. Autoradiography was for 2 hours.

FIG. 2 is a schematic diagram depicting the method of cloning of receptor or cytoplasmic tyrosine kinase targets (CORT), C-terminal domain of the EGFR is phosphorylated with radiolabelled phosphorous. Lambda gt11 library was plated at a density of 4×10⁴ plaques per 150 ml plate. The plaques were overlaid with IPTG-impregnated nitrocellulose filters for 12 hours, after which the plaques were transferred to nitrocellulose and incubated with the labelled probe. Positive colonies are then selected for further analysis.

FIGS. 3A and 3B show autoradiograms of phage expressing GRB-1 protein. A) Primary screen demonstrating one positive signal (arrow) out of 40,000 phage plated. B) Plaque purification of phage expressing GRB-1. All plaques bound to the (³² P)-labelled C-terminal domain of the EGFR.

FIGS. 4A to 4I show the DNA sequence and predicted amino acid sequence of GRB-1 (SEQ ID NOS:1-2; complementary strand is SEQ ID NO:48). The protein has 724 amino acid residues.

FIG. 5 compares the sequences of the SH2 domains of GRB-1 with other proteins with similar motifs. A) SH2 domains of GRB-1 (N=SEQ ID NO:17; C=SEQ ID NO:18), c-src (SEQ ID NO:19), v-abl (SEQ ID NO:20), bovine PLC-δ (N=SEQ ID NO:21; C=SEQ ID NO:22), GAP (N=SEQ ID NO:23; C=SEQ ID NO:24) and V-crk (SEQ ID NO:25). N and C refer to N-Terminal and C-terminal SH2 domains respectively. Conservation amino acid substitutions are as defined by Schwartz and Dayhoff: (A,G,P,S,T); (L,I,V,M); (D,E,N,Q); (K,R,H); (F,Y,W); and C. Bold letters identify those position were the same or a conservative amino acid substitution is present at 5 or more position. Boxes identify conserved motifs. B) A similar comparison of the SH3 domain of GRB-1 (SEQ ID NO:26); C-srk (SEQ ID NO:27); v-abl (SEQ ID NO:28); PLC (SEQ ID NO:29); GAP (SEQ ID NO:30); V-crk (SEQ ID NO:31).

FIG. 6 is a schematic diagram comparing the structural organization of the SH2 and SH3 domains. The scheme includes known proteins containing SH2 and SH3 domains, such as c-src, v-crk, PLC-δ, GAP1 and GRB-1.

FIG. 7 is a Northern blot of monkey mRNA with GRB-1 probe. 5 μg of poly (A)+MRNA, obtained from various monkey tissue, was electrophoresed on 1.2%/2.2M agarose-formaldehyde gel. The blot was hybridized with a (32P)-nick translated DNA probe corresponding to the insert from clone ki4.

FIG. 8 is a gel pattern showing that antibodies to GRB-1 immunoprecipitate a protein of 85 kDa from biosynthetically labelled cells. Cells were metabolically labelled with (³⁵ S)methionine, after which lysates were prepared and immunoprecipitated with either immune (I) or preimmune (P) serum. The immunoprecipitated protein was separated on a 8% SDS/PAGE. Autoradiography was performed overnight. Cell lines used include human glioblastoma cell line, U1242, rat bladder carcinoma cell line, NBT-II and NIH-3T3 cells.

FIGS. 9A and 9B depict several wild-type and mutant proteins used in the studies. (A) EGF receptor constructs with their known or predicted autophosphorylation sites. Wild-type (W.T.), Kinase negative (K721A), and carboxy-terminal deletion (CD126), were immunoprecipitated from previously described transfected NIH373 cells expressing -300,000 EGF receptors. EGFR-C represents a deletion mutant containing the cytoplasmic domain of the EGF receptor produced by baculovirus-infected SF9 cells. (B) Structure of PLC-δ and trpE/GAP SH2 proteins indicating location of the SH2 and SH3 domains and PLC-δ tyrosine phosphorylation sites.

FIG. 10A-10B is a gel pattern showing association of PLC-δ with EGFR mutants. Wild-type (HER14), carboxy-terminal deletion (DC126), or kinase-negative (K721A) EGFR were immunoprecipitated with anti-EGFR mAb108. Receptors were autophosphorylated with (δ-³² P-ATP. Concomitantly EGFR-C was added to protein A-Sepharose beads alone or to immunoprecipitated K721A receptors either with or without ATP. After further washes to remove ATP, lysate from -15×10 6 PLC-δ overexpressing 3T-P1 cells was added and mixed for 90 min at 4 C. After washing to remove unbound PLC-δ, proteins were separated on a 6% SDS-gel and transferred to nitrocellulose for immunoblotting. One eighth of the sample was utilized for anti-PTyr blotting, the reminder for anti-PLC-δ blotting (exposure time 14 h).

FIG. 11 is a gel pattern showing that phosphorylation of PLC-δ reduces its binding to the EGF receptor. Full length EGFR was immunoprecipitated with mAb108, and allowed to autophosphorylate. Lysate from PLC-δ overexpressing 3T-P1 cells was added and mixed for 90 min at 4° C. After binding, ATP was added to one half of the samples allowing the PLC-δ molecules to be phosphorylated by the EGF receptor. SDS-PAGE sample buffer was then added to one half of the EGFR-PLC-δ complexes (NO WASH, left panel) and directly loaded onto the 6% gel. The other half was washed three times with HNTG and then loaded on the gel (WASH, right panel. After running duplicate samples on SDS-PAGE, the proteins were transferred to nitrocellulose and probed with anti-PLC-δ and (¹²⁵ I)protein A. The bands were subsequently cut from the nitrocellulose and quantitated in a δ counter. After three washes with HNTG, 50±5% (Mean±SEM, n=4) of the non-phosphorylated PLC-δ remained bound to the EGFR while only 22±4% of the phosphorylated PLC-δ remained (exposure time: 12 h).

FIGS. 12A and 12B are representations of a gel pattern showing binding of EGFR-C to trpE proteins. In FIG. 12A, EGFR-C (0.5 μg) was immunoprecipitated with antibody C and washed. MnCl₂ alone or MnCl₂ and ATP were then added to facilitate autophosphorylation of TrpE or trpE/GAP SH2 (approximately 2 μg). The immunoprecipitates were separated on a 10% SDS-gel, transferred to nitrocellulose and immunoblotting was performed with anti-trpE. For comparison, about 0.1 μg of trpE or trpE/GAP SH2 lysate was loaded directly on to the gel (right panel of A). In FIG. 12B, trpE or trpE/GAP SH2 was immunoprecipitated with anti-trpE antibodies and washed. Phosphorylated or non-phosphorylated EGFR-C (0.5 μg) was then added and allowed to bind as above. After washing, samples were separated on a 10% gel, transferred to nitrocellulose and probed with antibody C. The two samples on the right represent 0.5 μg of phosphorylated and non-phosphorylated kinase loaded directly onto the gel (exposure time: 2 h).

FIGS. 13A and 13B are representations of a gel pattern showing binding of trpE/GAP SH2 to wild-type and mutant EGFR. In FIG. 13A, wild-type receptor (HER14) or the carboxy-terminal deletion CD126 receptor were immunoprecipitated with mAb 108. MnCl₂ alone or MnCl₂ and ATP were then added to the autophosphorylated half of the receptor-containing samples. One set of CD126 was also cross-phosphorylated with 0.5 μg of EGFR-C. TrpE/GAP SH2 was then added for 90 min at 4° C. and, after three more washes, loaded onto SDS-PAGE. After transfer to nitrocellulose, blots were probed with anti-trpE (left panel), anti-EGFR RK2 (center panel), or anti-PTyr (right panel). RK2 and anti-PTyr are both 1/8 of the total sample and were separated on 7% SDS-PAGE. The remaining sample was loaded on a 10% gel for the anti-trpE blot (exposure time 14 h).

In FIG. 13B, lysates from NIH3T3 2.2 cells containing no EGFR (3T3) or from cells with kinase-negative receptors (K21A) were immunoprecipitated with mAb108. To all immunoprecipitates, 0.5 μg of EGFR-C was added and then MnCl₂ alone or MnCl₂ and ATP. trpE/GAP SH2 was added and samples prepared and immunoblotted as in (A) (exposure time 19 h).

FIG. 14 is a gel pattern showing binding of PLC-δ and trpE/GAP SH2 to the CNBr cleaved C-terminal fragment of EGFR. EGFR-C (10 μg) was incubated in a Centricon 30 in 20 mM HEPES, pH 7.5 with 100 μg BSA as a carrier protein. The phosphorylated and non-phosphorylated EGFR-C were then each divided in two with one half being stored in buffer while the other half was cleaved with CNBr. The four samples either with or without ATP, and with or without CNBr were then each brought up in 500 μl 1% Triton X-100 lysis buffer, split in two, and immunoprecipitated with anti-C antibody. After washing the immunoprecipitates, lysates containing PLC-δ or trpE/GAP SH2 were added. Immunoblotting was then performed on the samples as above with anti-trpE or anti-PLC-δ. For the right panel, a fraction of the cleaved and uncleaved EGFR-C (0.1 μg) was loaded directly on the gel without immunoprecipitation and immunoblotted with RK2 (exposure time 14 h). The dark band seen in all lines of the anti-trpE blot runs at about 40 kDa (also seen in FIG. 13) and represents (¹²⁵ I)protein A binding to the heavy chain of the immunoprecipitating antibody.

FIG. 15 is a gel pattern showing binding of the tyrosine phosphorylated C-terminal EGFR fragment to trpE/GAP SH2 but not to trpE. EGFR-C (5 μg) was autophosphorylated by the addition of (δ-32P)ATP. The phosphorylated EGFR-C was concentrated in a Centricon 30, and then cleaved with CNBr in 70% formic acid. One half of the sample (350,000 c.p.m.) was allowed to bind to trpE or trpE/GAP SH2 as in FIG. 12B, washed and run on a 10% SDS-gel. (A) Binding of phosphorylated CNBr cleaved EGFR-C to trpE (B) Binding of phosphorylated CNBr cleaved EGFR-C to trpE GAP SH2 (C) 3000 c.p.m. of CNBr-cleaved EGFR-C (D) for comparison 3000 c.p.m. of cleaved EGFR-C (exposure time 20 h). EGFR 984/1186 indicates the sequence of the tyrosine autophosphorylated fragment generated by CNBr.

FIG. 16A-16D shows the partial nucleotide sequence (SEQ ID NO:32; complementary strand is SEQ ID NO:50) and predicted amino acid sequences (SEQ ID NOS:33-38) of GRB-2.

FIG. 17 is a comparison of sequence homology of avian crk (SEQ ID NO:39) to GRB-3 (SEQ ID NO:4) with dots indicating homologous amino acids.

FIG. 18 is a protein sequence of nck (SEQ ID NO:40) compared to that of GRB-4 (SEQ ID NO:8) for amino acid sequence homology.

FIG. 19 is a GRB-7 (SEQ ID NO:10) protein sequence.

FIG. 20 is a schematic representation of GRB-7 to include the proline rich, P2B2, rasGAP and SH2 domain homology.

FIG. 21 is a comparison of a GRB-7 amino acid sequences (SEQ ID NO:10) with SH2 domains from avian c-src (SEQ ID NO:19), human PLC-δ1(SEQ ID NO:22), GRB-1/p85 (SEQ ID NO:17), mouse fyn (SEQ ID NO:41), GRB-3 (SEQ ID NO:3) and GRB-4 (SEQ ID NO:8).

FIG. 22 is a comparison of a GRB-7 amino acid sequence (SEQ ID NO:10) with rasGAP (SEQ ID NO:42).

FIG. 23 is a comparison of a GRB-7 amino acid sequence (SEQ ID NO:10) with P2B2 (SEQ ID NO:43).

FIG. 24 is a representation of a Northern blot analysis of GRB-7 mRNA.

FIG. 25A-C is a comparison of binding of the phosphorylated EGFR carboxy-terminus to PLC-g fragments expressed in a λgt11 or T7 polymerase based library.

FIGS. 26A-26C include a cDNA (SEQ ID NO:5); complementary strand is (SEQ ID NO:49) and protein sequence (SEQ ID NO:6) of GRB2 clone 10-53, with '5 and '3 untranslated flanking sequences; SH2 (thick line) and SH3 (thin lines) domains are indicated.

FIG. 26D is a schematic representation of the overall domain structure of GRB2.

FIGS. 26E and 26F are sequence alignments of GRB2 SH2 (SEQ ID NO:44) and SH3 (N=SEQ ID NO:45; C=SEQ ID NO:46) domains, respectively, with other proteins. N and C refer to N-terminal and C-terminal domains, respectively. The one letter code is used to indicate amino acid residues. Bold letters identify those positions where the same or a conservative amino acid substitution is present at that position. Compared are PLCδ1, GAP, v-src, v-abl, v-crk and p85. The SH2 domain of GRB2 is most similar to the SH2 domain of v-fgr (43% similarity) and the N-terminal SH3 domain is most similar to the SH3 domain of human vav (48% similarity).

FIGS. 27A-27B show the analysis of expression of GRB2 in various murine tissues and cell lines. 27A shows a Northern analysis in murine tissues, with tissue of origin as indicated, with 20 μg total RNA loaded per lane. The sizes of the GRB2 transcripts (relative to BRL size markers indicated) are 3.8 kb and 1.5 kb.

FIG. 27B shows immunoprecipitation of GRB2 from (³⁵ S)methionine labeled HER14 lysates with preimmune (lane 1) and immune GRB2 antiserum (Ab50) (lane 2). Immunoblot analysis of GRB2 from lysates of HER14 cells with Ab86 (lane 3). Molecular weight markers (sized in kDa) are indicated. Arrow indicates band corresponding to GRB2 protein. Exposure times are 24 hours.

FIG. 28 shows the association of endogenous GRB2 with EGFR in HER14 cells. HER14 cells mock treated lanes 1, 3, 5) or EGF treated (lanes 2, 4, 6) were lysed and immunoprecipitated with anti-EGF receptor antibodies (mAb 108), subjected to SDS-PAGE, and after transfer to nitrocellulose, blotted with polyclonal anti-EGFR antibodies (Anti-C) lanes 1 and 2), anti-phosphotyrosine antibodies (lanes 3 and 4), or anti-GRB2 antibodies (Ab86) (lanes 5 and 6). The immunoblots were labeled with ¹²⁵ I-protein A followed by autoradiography at -70° C. Anti-GRB2 blot were exposed for 24 hrs. Anti-EGFR and antiP-tyr blots were exposed for 16 hrs. The positions of molecular weight markers (sized in kDa) are indicated.

FIG. 29 is a schematic representation of GRB2-GST fusion proteins. Gluthatione-S-transferase fusion proteins of full size GRB2 and various regions of GRB2 were generated and purified by affinity chromatography utilizing glutathione agarose beads, as described in methods. Shown are the SH2 domain of GRB2 (SH2), the amino terminal SH3 (N-SH3), carboxy terminal SH3 (C-SH3), the amino terminal SH3 and SH2 domains (N-SH3 SH2), and the SH domain with the carboxy terminal SH3 domain (SH2 C-SH3). GST region of fusion proteins is not shown.

FIG. 30 represents the binding of GST-GRB2 fusion proteins to activated growth factor receptors in vitro. Binding of fusion proteins to the tyrosine phosphorylated proteins (lanes 1 through 6) and EGFR (lanes 7 through 10) in control and EGF stimulated HER14 cell lysates, and tyrosine phosphorylated proteins in control and PDGF stimulated lysates (lanes 11 through 14). Lysates were incubated with equal amounts of fusion proteins immobilized on glutathione-agarose beads. Bound proteins were washed, subjected to SDS-PAGE and immunoblotted with antiphosphotyrosine (lanes 1 through 6, 11 through 14)) or anti EGF-receptor (lanes 7 through 10) antibodies. The immunoblots were labelled with ¹²⁵ I-proteins a followed by autoradiography at -70° C. exposure time 16 hrs. The positions of the molecular weight markers are indicated (sizes in kDA).

FIG. 31 shows data representing the lack of significant phosphorylation of GRB2 in HER14 cells following stimulation with EGF. (³² P)orthophosphate (lanes 1 through 4) or (³⁵ S) methionine (lanes 5 through 8) metabolically labeled HER14 cells were lysed following mocked EGF treatment. The precleared lysates were immunoprecipitated with either preimmune or anti-GRB2 antibodies (Ab50), and subjected to SDS-PAGE and autoradiography. Two hour (32P) and two day (³⁵ S) exposure times are shown. The position of GRB2 and the co-immunoprecipitating 55 kDa phosphoprotein are marked with arrows.

FIG. 32 presents the alignment of amino acid sequences of GRB2 (SEQ ID NO:6) and sem-5 (SEQ ID NO:44) (single letter code). Boxes surround the SH2 and SH3, domains, as indicated. Bold capital letters indicate identical amino acids, capital letter indicate conservative substitutions.

FIG. 33 is a representation showing a model for the interaction between EGF receptor and GRB2 and their C. elegans counterparts. Tyrosine autophosphorylated EGFR (or let-23) binds to the SH domain of GRB2 (or sem-5). Ras (or let-60) acts downstream leading to either cell proliferation or vulval development.

FIG. 34A-34C is a cDNA (SEQ ID NO:3) and protein sequence (SEQ ID NO:4) of GRB-3.

FIG. 35A-35B is a cDNA (SEQ ID NO:7) and protein (SEQ ID NO:8) sequence of GRB-4.

FIGS. 36A-36G is a cDNA (SEQ ID NO:9) and protein (SEQ ID NO:10) sequence of GRB-7.

DETAILED DESCRIPTION OF TEE PREFERRED EMBODIES

Methods, compounds and compositions have now been discovered to provide a means to understand and gain control over the regulation of cell growth and oncogenesis by providing the ability to identify target proteins for tyrosine kinases, including both receptor and cytoplasmic tyrosine kinases in eukaryotic organisms.

One embodiment of the present invention is to provide a novel expression/cloning system for the rapid cloning of target proteins which bind tyrosine kinase proteins which are present intracellularly and in cell receptors of eukaryotes. The cloning method is based on the discovery that certain class of substrates can bind specifically to the phosphorylated domain of proteins having tyrosine kinase activity.

According to another embodiment of the present invention, novel probes and methods using such probes for rapid expression cloning of DNA encoding proteins which have the characteristic of binding to the tyrosine-phosphorylated portion, such as the C-terminus, of a receptor tyrosine kinase molecule, which molecule is present in the cytoplasm or in cell receptors of eukaryotic receptors.

By the term "eukaryote" or "eurkaryotic" is intended any organism considered to have the attributes of a eukaryote, including a cell nucleus, mitochondria, chromosomes, etc., which are attributes which do not occur in bacteria, blue-green algae or viruses. Non-limiting examples of eukaryotes include yeast, fungi, insects, plants, mammals, birds, reptiles, amphibians. Mammals include, but are not limited to, humans, mice, rats, rabbits, cows, pigs, goats, sheep, horses, cats, dogs, etc.

Expression cloning is a method wherein the DNA being cloned encodes a protein which is expressed from a cloned library from a cell known or expected to have the desired protein. The desired DNA, typically in the form of a cDNA library, is detected by means of its expression and/or direct detection of the protein which it encodes. Expression cloning systems and library cloning are well-known in the art (see: Sambrook, J. et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al, eds. (Current Protocols in Molecular Biology Wiley Interscience, N.Y. (1987, 1992)), which references are hereby entirely incorporated by reference).

According to the present invention, the protein is expressed according to known method steps from a library and the expressed protein, released from the cell it is expressed in is transferred to a solid carrier or support, such as a nitrocellulose filter as a non-limiting example, and detected using a detectable label for the expressed protein by known method steps.

One of the ways in which the polypeptide probe target protein can be detectably labeled is by providing peptide probes or anti-target protein antibodies and linking the peptide probes or antibodies to an enzymefor use in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may additionally be accomplished using any of a variety of other immunoassays or detectably labeled peptide probes. For example, by radioactively labeling the peptide probes, anti-target protein antibodies or antibody fragments, such that the labeled target protein may also be detected through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S., et al., North Holland Publishing Company, New York (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by T. Chard, incorporated by reference herein. A radioactive isotope, such as ³² P, ³⁵ S, ¹² C or ³ H can be detected by such means as the use of a gamma counter, a liquid scintillation counter or by autoradiography.

It is also possible to label the peptide probe or anti-target protein antibody with a fluorescent compound. When the fluorescently labeled peptide or antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Suitable fluorescent probes are well known or commercially available, such as from Molecular Probes, Inc., Eugene Ore.

The peptide probe or anti-target protein antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵² Eu, or others of the lanthanide series. These metals can be attached to the peptide probe or anti-target protein antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The peptide probe or anti-target protein antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged peptide probe or anti-target protein antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the peptide probe or anti-target protein antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic peptide probe or anti-target protein antibody increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent peptide probe or anti-target protein antibody is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

The expression cloning method of the present invention for detecting and cloning a target protein for tyrosine kinase cytoplasmic or receptor protein may be used for detecting such target proteins from any eukaryotic cell source. For example, certain target molecules bind to the tyrosine phosphorylated portion of PDGFR and the colony stimulating factor-1 (CSF-1) (Coughlin, S. R. et al., Science 243:1191-1194 (1989); Kazlauskas, A. et al., Cell 58:1121-1133 (1989); Shurtleff, S. A. et al., EMBO J. 9:2415-2421 (1990); and Reedjik, M. et al., Mol. Cell. Biol. 10:5601-5608 (1990)). In these receptors, the tyrosine phosphorylation occurs in a kinase insert domain, rather than in the C-terminal domain as is the case with the EGFR. Therefore, specific polypeptide probes in the range of 10-250, such as 10-20, 20-30, 40-50, 70-100, or 100-200, amino acids utilizing the kinase insert domain, or a portion thereof as defined herein, and cytoplasmic or receptor or PDGFR or CSF-1 receptor can be similarly used for expression cloning. Similar probes can also be constructed for the fibroblast growth factor (FGF) receptor (which is tyrosine phosphorylated in the C-terminal domain) or the HER 2/neu receptor, both of the which are also able to interact with SH2 containing proteins such as PLC-δ. In other receptors, such as the insulin receptor, tyrosine phosphorylation occurs in the kinase domain itself.

Accordingly, any tyrosine kinase protein or fragment thereof of 10-250 amino acids, e.g., as described in Cantley et al. Cell 64:281-302 (1991) (the entire contents of which are herein incorporated by reference), can be used to bind a target protein in solution which is contacted to the tyrosine kinase protein bound or associated with a carrier or support. The carrier or support can be any known material that associates with a tyrosine kinase or fragment thereof, such that, once the target protein is bound, the non-bound material can be removed from the carrier without dissociated the tyrosine kinase bound to the target protein.

Thus the tyrosine kinase protein is used as a protein probe to bind target proteins. Alternatively, a polypeptide of 10-250 amino acids, corresponding to at least a phosphorylation domain of the tyrosine kinase; or corresponding to a consensus sequence of a class or group of tyrosine kinases, can be used as the protein or polypeptide probe and may be detectably labeled.

Thus, while it will be appreciated that different sites are tyrosine-phosphorylated in different proteins, e.g., the C-terminal domain in the EGFR, the kinase domain in insulin receptor, and a kinase domain insert in PDGFR, the present invention recognizes the common features of all these structures, the presence of one or more phosphotyrosine residues, and the ability of certain cellular proteins to bind on the basis of affinity to a polypeptide containing one or more phosphotyrosines. While reference will generally be made below to a probe which is a C-terminal domain, with reference to the EGFR, this language is not intended to be limiting and is intended to include all of the other alternative tyrosine-phosphorylated domains discussed above.

The methods and approach of the present invention can be applied to the cloning and identification of all target molecules which are capable of interacting in a specific manner with tyrosine phosphorylated polypeptides, such as cytoplasmic tyrosine kinases or the activated phosphorylated receptors described herein. Additional proteins which bind to tyrosine-phosphorylated sequences, such as the tyrosine-specific phosphatases, e.g., R-PTPases (Sap, J. et al., Proc. Natl. Acad. Sci. USA 87:6112-6116 (1990); Kaplan, R. et al., Proc. Natl. Acad. Sci. USA 87:7000-7004 (1990) may also be use according to a method of the present invention. The methods are also applicable in the cloning and identification of proteins which bind to phosphorylated serine/threonine residues, as with serine/threonine-specific phosphatases as a non-limiting example.

Use of a polypeptide or protein probe of the present invention allows the rapid cloning of DNA and identification of the encoded proteins from eukaryotic DNA or RNA libraries., such as a gene expression library. The method is particularly useful with a bacteriophage lambda gt11 library or a T7 library. As a non-limiting example of a eukaryotic library, screening a human fetal brain lambda gt11 expression library has permitted the present inventors to clone several target protein genes and to characterize the proteins they encode. One, termed GRB-1, was fully DNA sequenced (SEQ ID NO:1) and found to encode novel human protein with an amino acid sequence as shown in FIGS. 4A and 4B (SEQ ID NO:2) and a molecular weight of about 85 kDa which contained two SH2 domains and one SH3 domain (FIGS. 4A and 4B and FIG. 5). GRB-2 DNA (FIGS. 26A-26C) (SEQ ID NO:5) also contains unique SH2 and SH3 domains in the amino acid sequence, (FIGS 26A-26C) (SEQ ID NO:6). GRB-3 DNA (SEQ ID NO:3) was also sequenced (FIGS. 34A-34C) and the GRB-3 amino acid sequence (SEQ ID NO:4). GRB-4 DNA (SEQ ID NO:7) (FIGS. 35A-35B) encoded a protein composed of three SH3 domains and one SH2 domain having the GRB-4 amino acid sequence (SEQ ID NO:8).

Several overlapping clones were identified which were used for DNA sequencing of GRB-7 (FIGS. 36A-36G) (SEQ ID NO:9) to obtain the full length GRB-7 amino acid sequence shown in FIGS. 36A-36G (SEQ ID NO:10). A schematic representation of GRB-7 is displayed in FIG. 20 depicting the regions of similarity to known proteins. The GRB-7 protein is 535 amino acids in length (FIGS. 36A-36G) (SEQ ID NO:10) and has one SH2 domain at its extreme carboxy-terminus. In FIG. 21, the SH2 domain of GRB-7 is compared to other SH2 domains including mouse fyn, human PLC-δ1 and the crk and nck-like proteins of the present invention. Other protein motifs in GRB-7 were determined using Swissprot and GenEmbl databases, using software such as the University of Wisconsin Genetics Computer Group Sequence Analysis Software package (Devereaux et al Nucl. Acid Res. 12:387 (1984)). The Swissprot and GenEMBL database can be searched using known software, such as the FASTA and TFASTA respectively. Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988). Protein alignments can be performed using known software, such as BESTFIT, e.g., with conservative substitutions defined as a score of ≧0.8 using the symbol comparison table for proteins. Gribskov and Burgess, Nucleic Acid Research 14, 6745 (1984).

From such analysis, amino acids 242 to 339 of GRB-7 showed similarity to a sequence from the central region of ras GAP (FIG. 21). Over this region of 91 amino acids from ras GAP, GRB-7 has 26% identity and 42% similarity allowing for conservative substitutions (FIG. 22). This region of ras GAP lies between the SH2/SH3 domains and the GTPase activating carboxy terminal region and has not been assigned a specific function (Martin et al Science 255:192 (1992)). The amino-terminal sequence of GRB-7 was found to be proline rich and thus has similarity to many other proline rich proteins. GRB-7 does have an extended region of limited similarity to the catalytic domain of protein phosphatase 2B (Guerini and Klee, Proc. Natl. Acad. Sci. USA 87:6112 (1990)) including this proline rich region (FIG. 23) but no significant similarity was found to other serine/threonine phosphatase such as protein phosphatase 1 or 2A.

A northern blot of GRB-7 in mouse tissues is presented in FIG. 24. Oligo dt selected mRNA was probed with GRB-7 cDNA using known methods. See Ausubel et al eds., Current Protocols in Molecular Biology, Wiley Interscience, New York, (1987, 1992) and Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990), which are entirely incorporated herein by reference. The highest signal was detected in liver and kidney, but a signal was also detected in ovary and testes. On longer exposure, a weak signal was detectable in lung but not in heart, muscle, spleen or brain. The major transcript was seen at 2.4 kb which closely corresponds to the longest cDNA clone obtained.

GRB-7 represents another novel gene cloned using the CORT technology, according to the present invention. It belongs to a relatively rare group of proteins with SH2 domains but no SH3 domains including the fps tyrosine kinase, (I. Sadowski, J. C. Stone and T. Pawson, Mol. Cell. Biol. 6:4396 (1986)), protein tyrosine phosphatase 1C (Shen et al Nature (Lond.) 352:736 (1991)) and possibly tensin (Davis et al., Science 252:712 (1991)).

CORT methodology of the present invention provides proteins that interact with the EGFR and lie downstream of the EGFR signalling pathway. In general, in vitro associations between SH2 domain and tyrosine phosphorylated proteins correlate with interactions in living cells (McGlade et al., Mol. Cell. Biol. 12:991 (1992)). CORT methodology of the present invention is therefore expected to yield commercially important downstream signalling components of cytoplasmic tyrosine kinase target proteins, as well as growth factor receptors, as demonstrated by the finding that the C. elegans gene sem-5 is the homolog of human GRB-2. Sem-5 is crucial for vulval development, a process that requires the activity of let-23, an EGFR like tyrosine kinase. Accordingly, it is expected that sem-5 lies downstream of the activated let-23, and that GRB-2 serves a similar crucial function in EGFR signalling.

CORT methodology of the present invention can also be used to identify new SH2 proteins that interact with the EGFR. Seven different exemplary SH2 domain proteins are expected to have important signalling functions. With the use of the T7 polymerase based library, this methodology may be more easily applied, due to relatively higher levels of expressions which increase detectability, to any eukaryotic cytoplasmic or receptor tyrosine kinase proteins, such as growth factor receptor systems. Hence such a method of the present invention can also be used to clone other novel SH2 domain proteins using other growth factor receptor tyrosine kinases, including the use of T7 polymerase based libraries, by performing expression/cloning techniques involving protein-protein interactions and DNA binding proteins.

SH2 domains, such as in the GAP and PLC-δ proteins, are responsible for the association of these proteins with the phosphorylated C-terminus of the EGFR (see Example VI, below). Thus, one function of SH2 domains is to juxtapose the intracellular portion of receptor tyrosine kinase molecules with their substrates to facilitate efficient tyrosine phosphorylation.

Detailed analysis of one of the cDNA clones of the present invention, GRB-1, identified using methods of the present invention, reveals a novel sequence containing two SH2 domains and one SH3 domain. This protein is expressed in various tissues and cell lines. Its predicted molecular weight, 85 kDa, is consistent with its migration on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

By the term "cytoplasmic tyrosine kinase" is meant a soluble form of proteins or polypeptide having tyrosine kinase which can be found in the intracellular portion of a cell. By the term "receptor tyrosine kinase" is intended a transmembrane protein having an extracellular receptor domain, and one or more intracellular domains, including at least one extracellular or intracellular domain having tyrosine kinase enzymatic activity. Additional intracellular domains may have sequence homology to SH2. These molecules are well known in the art (Williams, L. T. et al., Science 243:1564-1570 (1989); Ullrich, A. et al., Cell 61:203-212 (1990); Carpenter, G. et al. J. Biol. Chem. 265: 7709-7712 (1990), which are entirely incorporated by reference).

The proteins which interact with, and which may be phosphorylated by, tyrosine kinases are referred to as "target" proteins for these kinases, as distinguished from the "ligands" for these receptors, which bind to the kinase.

According to the present invention, an expression cloning method is performed directly on a gene expression library, such as lambda gt11 or T7 expression library. In a preferred embodiment, the DNA is human cDNA. More preferably, the DNA is human fetal brain DNA. Using such a source as the starting material for the cloning of human genes has a great advantage over the alternative known means, in which a large amount of tissue is taken, and antibodies produced, or the protein purified and partially sequenced, and oligonucleotide probes are then prepared from this sequence and used to screen a genomic DNA or cDNA library. The advantage of bypassing these steps is of most relevance in the case of human genes, since tissue is generally not available in large quantities, with the exception of placenta.

The expression library may be screened in a single step. Preferably, the lambda plaques are blotted onto a solid carrier, preferably nitrocellulose, allowing the transfer of library DNA-encoded proteins which are expressed in the infected bacteria and transferred to the carrier. This carrier is then incubated with the probe of the present invention, as described herein. The probe is allowed to bind to proteins which have the capability of binding to the tyrosine-phosphorylated polypeptide. Based on the label used in the probe, such as an enzymatic, radioisotope or fluorescent label, an appropriate detection system is used to identify the plaques containing the protein of interest. The phage in these plaques are then selected, and the DNA inserts can then be re-cloned, excised and placed into other vectors, used for large scale expression of the protein, and the like, according to known method steps.

One of ordinary skill in the art will appreciate that the concentrations, times, temperatures can be varied depending on the precise nature of the system used, and will know how to vary the appropriate parameters without undue experimentation. Furthermore, general methods in this area are set forth in Sambrook et al. (supra).

Materials of which solid phase carrier can be made include, but are not limited to, nitrocellulose, cellulose, paper, substituted polystyrenes, acrylonitriles, polycarbonate, polypetene, or silicone oxide.

The probe of the present invention is a tyrosine-phosphorylated polypeptide molecule derived from the C-terminal domain of a cytoplasmic or receptor tyrosine kinase. The polypeptide can have between about 10 and about 250 amino acids in length. The probe can be a phosphorylated native sequence or a functional derivative thereof (defined below).

Highly efficient phosphorylation is obtained by using the tyrosine kinase domain present on the tyrosine kinase molecule to autophosphorylate the C-terminal region at between 1 and 5 tyrosine residues. Known methods and conditions (described in detail in Example I) are used to phosphorylate the tyrosine residues. A preferred substrate is detectably labeled substrate such as (δ-P³² -adenosine triphosphate). The source of tyrosine molecule used as the source material to make the probe can include molecules chemically purified from tissues or cells, or molecules produced recombinant DNA methods.

When using recombinant techniques, a native cytoplasmic or receptor tyrosine kinase may be produced, or alternatively, a tyrosine kinase derivative may be produced. A preferred tyrosine kinase derivative includes the tyrosine kinase domain linked to the C-terminal domain. In another embodiment, the two domains may be produced as separate molecules, and mixed together to achieve tyrosine phosphorylation of the C-terminus-derived polypeptide.

The probe comprising a tyrosine-phosphorylated C-terminal portion of the tyrosine kinase, as described herein can be produced by recombinant means in the form of a fusion protein.

As used herein, a "fusion protein" may refer to a fused protein comprising a bacterial protein and a polypeptide of interest such as a protein having an SH2 domain. Alternatively, a fusion protein may also be an artificially constructed tyrosine kinase-like derivative, wherein a DNA sequence encoding the tyrosine kinase domain has been linked to a selective enzymatic cleavage site, which, in turn, is linked to a tyrosine kinase C-terminal domain having one or more tyrosine residues which can be phosphorylated by the kinase. Such a genetic construct encoding this type of "fusion protein" can be inserted into an expression vehicle and expressed in a bacterial or eukaryotic host. Once expressed, such a fusion protein can be allowed to autophosphorylate, wherein the kinase acts to phosphorylate the tyrosine residues in the C-terminal domain. Following this phosphorylation, use of the appropriate enzyme will cleave at the selective cleavage site, thus separating the N-terminal kinase from the C-terminal phosphorylated polypeptide, which can now serve as a probe.

Expression of fusion proteins and modifications to increase yields ans to provide cleavage sites, etc., are well known. See, e.g., Ausubel, supra; Itakura et al. Science 198:1056-1063 (1977)) and Riggs (U.S. Pat. No. 4,366,246 (1982); Marston, Biochem. J. 240:1-12 (1986); Nagai et al. (Nature 309:810-812 (1984); (Germino et al., Proc. Natl. Acad. Sci. USA 81:692-4696 (1984); Scholtissek et al., Gene 62:55-64 (1988); Smith et al., Gene 67:31-40 (1988); Knott et al., Eur. J. Biochem. 174:405-410 (1988); and Dykes et al., Eur. J. Biochem. 174:411-416 (1988), which references are all entirely incorporated herein by reference.

The term "selective cleavage site" refers to an amino acid residue or residues which can be selectively cleaved with either chemicals or enzymes and where cleavage can be achieved in a predictable manner. A selective enzymatic cleavage site is an amino acid or a peptide sequence which is recognized and hydrolyzed by a proteolytic enzyme. Examples of such sites include trypsin or chymotrypsin cleavage sites. In a preferred embodiment of this invention, the selective cleavage site is comprised of the sequence Ile-Glu-Gly-Arg (SEQ ID NO: 15), which is recognized and cleaved by blood coagulation factor Xa. In another embodiment, the selective cleavage site has the sequence Leu-Val-Pro-Arg (SEQ ID NO:16), which is recognized and cleaved by thrombin.

In constructing the tyrosine kinase-like derivative, an oligonucleotide sequence, 5' to the sequence coding for the enzyme recognition site can be included, and may vary in length. For example, in one embodiment, 13 nucleotides are situated between the codon for Ile (the start of the factor Xa recognition site) and the 3' end of the sequence encoding the tyrosine kinase domain.

Thus, in one embodiment of the present invention, the Ile-Glu-Gly-Arg (SEQ ID NO:15) sequence is introduced between the tyrosine kinase domain and the C-terminal domain. In another embodiment, the Leu-Val-Pro-Arg (SEQ ID NO:16) sequence is introduced. The proteins having this cleavage site are expressed in bacteria using standard methods. Thereafter, autophosphorylation of the C-terminal domain, preferably with (δ32P) adenosine triphosphate, is allowed to occur, followed by selective cleavage of the tyrosine-phosphorylated C-terminal domain with the appropriate cleaving agent, e.g., factor Xa.

The present invention also provides a method for mapping a gene, preferably a human gene, which encodes a target protein for a tyrosine kinase (such as a GRB protein as defined herein), to a particular human chromosome. This method combines the new expression cloning method described herein with one of several known techniques for mapping a gene to a particular chromosome. Thus, according to the present invention, a clone, such as a lambda gt11 clone, containing a DNA insert encoding a GRB protein, is identified using the expression cloning methods disclosed herein. The insert may be further subcloned, if desired, using methods well-known in the art, and a probe constructed, either by direct labeling of the nucleic acid of the clone or by producing an oligonucleotide probe corresponding to a unique portion of the clone's sequence (see: Sambrook, J. et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); and Ausubel, supra). This labeled probe is then used in a hybridization assay with commercially available blots, such Chromosome Blots from Bios Corporation (New Haven, Conn.) which contain DNA from a panel of human-hamster somatic cell hybrids (Kouri, R. E. et al., Cytogenet. Cell Genet. 51:1025 (1989)). By comparison of which human chromosomes remain in the human-hamster hybrid cell and the hybridization of the probe specific for the GRB gene of interest, the gene is mapped to a particular human chromosome. In this way, linkage is established to known human genes (or diseases caused by mutations therein) present on this chromosome. Using methods well-known in the art for finer mapping, e.g., using known human deletion mutations, the GRB gene can be mapped more precisely to other human genes.

The tyrosine-phosphorylated tyrosine kinase C-terminal probe polypeptide of the present invention, as well as the GRB proteins of the present invention, and additional yet unknown GRB proteins which are discovered using the methods of this invention, are useful in methods for screening drugs and other agents which are capable of modulating cell growth control that occurs via signal transduction through tyrosine kinases. By attaching a tyrosine-phosphorylated probe polypeptide or a GRB protein, or fragments thereof, to a solid phase carrier matrix, an affinity probe is created which can be used to isolate and purify molecules from complex mixtures which are capable of binding to the affinity probe. Furthermore, such an affinity probe is useful for detecting the presence in a biological fluid of a molecule capable of binding the tyrosine-phosphorylated probe or the GRB protein. Similarly, chemical agents can be tested for their capacity to interact with the probe or GRB.

Methods for coupling proteins and peptides to the solid phase, the solid phase substances useful in these methods, and means for elution, are well known to those of skill in the art.

In the case of growth factor receptors which are receptor tyrosine kinases (including as non-limiting examples EDGFR, PDGFR and FGFR), tyrosine phosphorylation is linked to cell growth and to oncogenic transformation. Disruption of the action of a GRB in the cell may prevent or inhibit growth, and might serve as means to counteract development of a tumor. Furthermore, a mutation in the C-terminal portion of the tyrosine kinase or the GRB, or a disregulation in their mutual interactions, may promote susceptibility to cancer.

The insulin receptor (InsR) is also a receptor tyrosine kinase, and tyrosine phosphorylation in cells bearing InsR is associated with normal physiological function. In contrast to the case of cell growth and cancer, disruption of normal interactions between of the tyrosine-phosphorylated portion of the receptor and the GRB would counteract insulin effects. Subnormal levels or activity of a GRB protein may act to remove a normal counterregulatory mechanisms. It is expected that overexpression or overactivity of a GRB protein could inhibit or totally prevent the action of insulin on cells, leading to diabetes (of an insulin-resistant variety). Thus susceptibility to diabetes may be associated with GRB protein dysregulation.

Therefore methods of the present invention for identifying normal or mutant GRB protein genes, or for detecting the presence or the amount of GRB protein in a cell, can serve as methods for identifying susceptibility to cancer, diabetes, or other diseases associated with alterations in cellular metabolism mediated by tyrosine kinase pathways.

The present invention provides methods for evaluating the presence, and the level of normal or mutant GRB protein in a subject. Altered expression of these proteins, or presence of a mutant GRB protein, in an individual may serve as an important predictor of susceptibility to oncogenic transformation and the development of cancer. Alternatively, altered expression of GRB protein may serve as an important predictor of susceptibility to diabetes.

Oligonucleotide probes encoding various portions of the GRB protein are used to test cells from a subject for the presence DNA or RNA sequences encoding the GRB protein. A preferred probe would be one directed to the nucleic acid sequence encoding at least 4 amino acid residues, and preferably at least 5 amino acid residues of the GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7, protein of the present invention, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids. Qualitative or quantitative assays can be performed using such probes. For example, Northern analysis (see Example III, below) is used to measure expression of an GRB protein mRNA in a cell or tissue preparation.

Such methods can be used even with very small amounts of DNA obtained from an individual, following use of selective amplification techniques. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Sambrook et al. (supra), Ausubel et al, supra, etc.

Recently, an in vitro, enzymatic method has been described which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been referred to as the "polymerase chain reaction or "PCR" (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194; Mullis, K. B. (Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986)); Saiki, R. K., et al, (Bio/Technology 3:1008-1012 (1985)); and Mullis, K. B., et al. (Meth. Enzymol. 155:335-350 (1987), which references are entirely incorporated herein by reference).

In one embodiment, the invention is directed to target proteins of eukaryotic tyrosine kinases, which include, as non-limiting examples, GRB proteins such as GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 proteins are included. In another embodiment, the invention is directed to recombinant eukaryotic GRB proteins. The invention provides the naturally occurring protein molecule substantially free of other proteins with which it is natively associated. "Substantially free of other proteins or glycoproteins" indicates that the protein has been purified away from at least 90 per cent (on a weight basis), and from even at least 99 per cent if desired, of other proteins and glycoproteins with which it is natively associated, and is therefore substantially free of them. That can be achieved by subjecting the cells, tissue or fluids containing the GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 protein to standard protein purification techniques such as immunoadsorbent columns bearing monoclonal antibodies reactive against the protein.

The nucleotide sequence of the GRB-1 gene (SEQ ID NO:1), and the amino acid sequence of the GRB-1 protein (SEQ ID NO:2), are shown in FIGS. 4A to 4I (SEQ ID NO:5). The partial nucleotide sequence of GRB-2 (1-949 of SEQ ID NO:32) and the partial amino acid sequence, are shown in FIGS. 16A-16D, and the complete amino acid sequence is shown in FIGS. 26A-26C (SEQ ID NO:6), as well as the complete nucleotide sequence.

In a preferred embodiment, GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7, or other eukaryotic GRB protein, can be isolated and purified using as an affinity probe, the probe of the present invention which is a tyrosine-phosphorylated C-terminal domain of a tyrosine kinase, or a functional derivative thereof.

Alternatively, the purification can be achieved by a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography, and ion exchange chromatography.

It will be understood that the GRB-1 proteins of the present invention can be biochemically purified from a variety of cell or tissue sources. For preparation of naturally occurring GRB protein, tissues such as mammalian placenta or brain are preferred.

Alternatively, because the gene for GRB1, GRB-2, GRB-3, GRB-4 or GRB-7 can be isolated or synthesized, the polypeptide can be synthesized substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic organism or in a non-mammalian eukaryotic organism, if desired. As intended by the present invention, a recombinant GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 molecule produced in mammalian cells, such as transfected COS, NIH-3T3, or CHO cells, for example, is either a naturally occurring protein sequence or a functional derivative thereof. Where a naturally occurring protein or glycoprotein is produced by recombinant means, it is provided substantially free of the other proteins and glycoproteins with which it is natively associated.

Alternatively, methods are well known for the synthesis of polypeptides of desired sequence on solid phase supports and their subsequent separation from the support or carrier. In particular, the tyrosine-phosphorylated C-terminal domain probe of the present invention, or a functional derivative thereof, can be synthesized using a peptide synthesis method wherein phosphotyrosine is provided in place of tyrosine, resulting in direct synthesis of the phosphorylated form of the polypeptide. See, e.g., Staerkaer et al, Tetrahedron Letters 32:5289-5392 (1991); Shoelson et al Tetrahedron Letters 32:6061 (1991), which references are entirely incorporated herein by reference).

The present invention also provides "functional derivatives" of the tyrosine-phosphorylated C-terminal domain polypeptide and or the GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 proteins.

By "functional derivative" is meant a "fragment," "variant," "analog," or "chemical derivative" of the GRB protein, which terms are defined below. A functional derivative retains at least a portion of the function of the native protein which permits its utility in accordance with the present invention.

A "fragment" of any of the proteins or polypeptides of the present invention refers to any subset of the molecule, that is, a shorter peptide.

A "variant" of the protein refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well-known in the art.

The term "substantially corresponding to the amino acid sequence of" in the context of the present refers to a protein containing conservative amino acid substitutions, known in the art and as described herein, that would be expected to maintain the functional biological activity of the referenced sequence, and/or target protein binding characteristics.

Such substitutions can be readily determined without undue experimentation by using known conservative substitutions, as known in the art. Alternatively, known software can be used to provide such conservative substitutions according to the present invention. As a non-limiting example the program "BESTFIT" can be used to provide conservative amino acid substitutions of a define sequence, e.g., defined as having a score of ≧0.4, 0.6, 0.8 or 1.0 depending on the type of protein used. See e.g., Gribskov and Burgess, Nucl. Acid. Res. 14:6745 (1984), which is entirely incorporated by reference. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide using methods well-known in the art.

Alternatively, amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see European Patent Publication No. EP 75,444).

At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis (as exemplified by Adelman et al., DNA 2:183 (1983)) of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture (see below). The variants typically exhibit the same qualitative biological activity as the nonvariant peptide.

Amino acid substitutions in the context of the present invention include substitutions wherein at least one amino acid residue in the peptide molecule, and preferably, only one, has been removed and a different residue inserted in its place. For a detailed description of protein chemistry and structure, see Schulz, G. E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins: Structure and Molecule Properties, W. H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. The types of substitutions which may by made in the protein or peptide molecule of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schultz et al. (supra) and FIGS. 3-9 of Creighton (supra). Base on such an analysis, conservative substitutions are defined herein as exchanges within one of the following five groups:

1. Small aliphatic, nonpolar or slightly polar residues: ala, ser, thr (pro, gly);

2. Polar, negatively charged residues and their amides: asp, asn, glu, gly;

3. Polar, positively charged residues: his, arg, lys;

4. Large aliphatic, nonpolar residues: Met, leu, ile, val (cys); and

5. Large aromatic residues: phe, tyr, trp.

Accordingly, amino acid sequences substantially corresponding to a given sequence can be made without undue experimentation and then routinely screened for tyrosine kinase binding activity using known methods or those disclosed herein, such that one of ordinary skill in the art can determine which substitutions provide tyrosine kinase target proteins according to the present invention. For example, once target protein sequences are determined, such as for GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7, conservative amino acid substitutions can be made to provide target proteins having amino acid sequences which substantially correspond to the determined target protein sequences.

The preferred bacterial host for this invention is E. coli. In other embodiments, other bacterial species can be used. In yet other embodiments, eukaryotic cells may be utilized, such as, for example, yeast, filamentous fungi, or the like. Use of these cell types are well known in the art. Any host may be used to express the protein which is compatible with replicon and control sequences in the expression plasmid. In general, vectors containing replicon and control sequences are derived from species compatible with a host cell are used in connection with the host. The vector ordinarily carries a replicon site, as well as specific genes which are capable of providing phenotypic selection in infected or in transformed cells. The expression of the fusion protein can also be placed under control with other regulatory sequences which may be homologous to the organism in its untransformed state. Preferred promoters can include a T7 promoter. Such preferred promoters express the human gene as a fusion protein such as the T7 capsid protein P10 under control of the T7 promoter. Such expression systems are commercially available, e.g, as the λEXlox vector from Novagen, Inc. (Madison, Wis.). In such fusion protein expression systems, the recombinant T7 vector containing a human gene, encoding such proteins obtainable by methods of the present invention, such as GRB-1, GRB-2, GRB-3, GRB-4 and GRB-7, as, e.g., a T10 fusion protein. The recombinant T7 vector can then be used to transform a bacteria, such as E. coli, by infection with a phage containing the recombinant T7 vector under lac control, such lacUV5 control. Induction of the infected, successfully transformed bacteria or other suitable host cell, by IPTG generates the T7 polymerase which then initiates transcription of the fusion protein encoded by the phage library. Because such resulting T7 vector infected bacteria provide human gene library plaques that have stronger signals than obtained by the use of bacterial RNA polymerases, such as E. coli RNA polymerase. According to the present invention, the use of a T7 polymerase expression system is particularly suitable for library screening when there as thousands of small plaques per plate. The major advantage of the use of a T7 expression System is the high level of protein expression due to the greater activity of the T7 polymerase versus E. coli RNA polymerase, and because fusion proteins using the smaller phage fusion protein gene, such as the T10 gene fragment (26 kd versus the 110 kd B-galactosidase of λgt11 expression library) yields more stable expression and that its hydrophobic character promotes binding to nitrocellulose. In addition to directional cloning, the use of T7 phages also allow for automatic conversion to a PET plasmid (see, e.g., Palazzalo et al., Gene 88, 25 (1990)) which can be useful for expression of a fusion protein for antibody production.

This invention is also directed to an antibody specific for an epitope of the GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 protein and the use of such an antibody to detect the presence of, or measure the quantity or concentration of, the GRB protein in a cell, a cell or tissue extract, or a biological fluid.

The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and anti-idiotypic (anti-Id) antibodies.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.

Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. The hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo production makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane-primed BALB/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl, Acad, Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published Nov. 14, 1984); Neuberger et al., Nature 314:268-270 (1985); Taniguchi et al., European Patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO 86/01533, (published Mar. 13, 1986); Kudo et al., European Patent Application 184187 (published Jun. 11, 1986); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986); Robinson et al., International Patent Publication #PCT/US86/02269 (published 7 May 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988); and Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988)). These references are hereby entirely incorporated by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody).

The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity.

Accordingly, mAbs generated against the GRB protein of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for a GRB protein epitope.

The anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as GRB protein-α.

The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')₂, which are capable of binding antigen. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab')₂ and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of GRB protein according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments).

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells which express the GRB protein. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.

The antibodies (of fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of GRB proteins. In situ detection may be accomplished by removing a histological specimen form a patient, and providing the a labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the GRB protein but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Such assays for GRB protein typically comprises incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying GRB protein, and detecting the antibody by any of a number of techniques well-known in the art.

The biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers followed by treatment with the detectably labeled GRB protein-specific antibody. The solid phase support or carrier my then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by conventional means.

By "solid phase support", "solid phase carrier", "solid support", "solid carrier", "support" or "carrier" is intended any support or carrier capable of binding antigen or antibodies. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrier configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-GRB-1, anti-GRB-2, anti-GRB-3, Anti-GRB-4 or anti-GRB-7, antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.

One of the ways in which a GRB-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactivity labeling the antibodies or antibody fragments, it is possible to detect R-PTPase through the use of a radioimmunoassay (RIA). A good description of RIA maybe found in Laboratory Techniques and Bio chemistry in Molecular Biology, by Work, T. S. et al., North Holland Publishing Company, N.Y. (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a δ counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can be then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵² EU, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

The antibody molecules of the present invention may be adapted for utilization in a immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen form the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" assay, which my also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.

The following example are presented by way of further explanation of the present invention, and not by way of limitation.

EXAMPLE I

A study was performed to determine the detectability of binding of the C-terminal domain of EGFR to a protein containing the SH2 domain immobilized on nitrocellulose filters. For this purpose, the binding of the C-terminal domain to a bacterially expressed fusion protein was assessed (see FIG. 1).

A. Isolation and Labelling of the Carboxyterminal Domain of the EGFR

The intracellular portion of the EGFR, which includes the tyrosine kinase domain and the carboxy terminal domain, was purified from recombinant baculovirus which expressed cDNA complementary to the intracellular domain of the human EGFR, as described previously (Hsu, C-Y. et al., Cell Growth and Differentiation 1:191-200 (1990)). The recombinant protein (2 μg) was then phosphorylated with (δ-³² P)ATP (200 μCi, 6000 Ci/Mmol)., at 4° C. in HNTG (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol) buffer which contained 5 mM MnCl₂. In order to remove unincorporated (y-32P) ATP, the phosphorylated kinase was diluted to 1 ml with 20 mM HEPES, pH 7.5, containing 100 μg BSA and then concentrated in a Centricon-10 to a volume of 50 μl. This procedure was repeated 3 times resulting in the removal of >99% of the unincorporated ATP. To separate the C-terminal domain from the kinase domain, the concentrated protein was then digested with cyanogen bromide (CNBr) in 70% formic acid for 14 hours at room temperature (see also Example VI, below). Samples were then washed three times with water, dried and resuspended in binding buffer to a concentration of 2×10⁶ cpm/ml.

B. Binding of the C-terminal Domain of the EGFR to Bacterially Expressed TrpE/GAP-SH2 Fusion Protein Immobilized on Nitrocellulose

TrpE and TrpE/GAP-SH2 were obtained from the laboratory of Dr. Tony Pawson and/or prepared as previously described (Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990)). Filter binding studies were performed according to published methods (Schneider, W. J. et al., Proc. Natl. Acad. Sci. 76:5577-5581 (1979); Daniel, T. O. et al., J. Biol. Chem. 258:4606-4611 (1983)) with minor modifications. Various concentrations of either bacterially expressed TrpE fusion protein or bacterial protein alone were spotted onto nitrocellulose filters. After blocking the filters for 1 hour at 4° C. in PBS containing 5% Carnation dry milk, ₃₂ P-labelled C-terminal domain of the EGFR was added and incubation was continued overnight at 4° C. After 24 hours, the nitrocellulose filters were washed 3 times at room temperature with PBS containing 0.2% Triton X-100. The filters were dried and exposed to Kodak XAR-5 film at -80° C.

C. Results

The above method permitted detection of specific binding of the EGFR C-terminal domain to less than 5 ng of a bacterially expressed GAP-SH2 fusion protein. The binding was specific, since it required tyrosine phosphorylation of the probe and did not occur when irrelevant proteins were applied to nitrocellulose filters.

The demonstration that the EGFR C-terminal domain could bind specifically to an SH2-containing protein immobilized on nitrocellulose filters encouraged the present inventors to apply this approach to the screening of lambda gt11 expression libraries with the goal of identifying novel EGFR binding proteins.

EXAMPLE II Screening of Expression Libraries and Isolation of a cDNA Clone Encoding a Novel SH2-Containing Protein

The tyrosine phosphorylated C-terminal tail of the EGFR was used as a probe to screen expression libraries from several different human tissues as described above. The approach to screening is outlined in FIG. 2. Numerous positive clones have been identified so far using this approach, of which two have been analyzed in detail.

A. Screening of cDNA Library

A lambda gt11, library, constructed from mRNA isolated from human brain stem, was obtained from M. Jaye. To screen the library, lambda gt11 phage were plated at a density sufficient to produce 4×10⁴ plaques per 150 mm agar plate. A total of six plates were initially screened. After incubation of the plates for 4 hours at 42° C., the plates were overlaid with nitrocellulose filters which had been impregnated with isopropyl-B-D-thiogalactopyranoside (IPTG), as previously described (MacGregor, P. F. et al., Oncogene 5:451-458 (1990)). Incubation was continued overnight at 37° C. The filters were then removed, washed with tBST (10 mM Tris-HCl, pH8, 150 mM NaCl, and 0.05% triton X-100) at room temperature, and then blocked in HBB (20 mM HEPES, pH 7.5, 5 mM Mg/Cl, 1 mM KCl) buffer containing 5% carnation dry milk for 1 hour at 4° C., as described (MacGregor et al., supra). Following blocking, labelled tyrosine phosphorylated carboxy-terminus (C-terminus) probe was added at a concentration of 1.6×10-4 μ/ml, and incubation was continued overnight. The filters were then washed 3 times at room temperature in PBS containing 0.2% Triton X-100. Filters were dried and exposed to Kodak XAR-5 film at -80° C.

Agar plugs, corresponding to the positive clones, were collect from the plates and placed in 1 ml of SM media. After allowing the phases to diffuse from the agar, the phases were replated and rescreened as described above. Those phages that demonstrated enrichment on subsequent screening were isolated and sequence. Lambda gt11 phage DNA was isolated by the plate lysate method according to Maniatis et al., and subcloned into EcoRI-digested M13 MP19 (Maniatis et al., 1982). Single stranded DNA was isolated and sequenced by the dideoxy chain termination method using the Sequenase DNA sequencing kit (United States Biochemical).

In one experiment, 240,000 pfu from a human brainstem lambda gt11 library were screened. A single plaque, clone ki4 (FIG. 3A) was isolated. On subsequent screening this clone demonstrated enrichment, and on tertiary screening all plaques bound the probe (FIG. 3B). Clone ki4 contained an insert of about 900 nucleotides, which, upon induction of the lac promoter with IPTG, produced a fusion protein which could bind the EGFR. The size of the fusion protein predicted that the cDNA insert coded for a protein of about 300 amino acids, which was the size expected if the cDNA contained a single large open reading frame. To analyze clone ki4 in more detail, DNA was isolated and the EcoRI fragment, corresponding to the human cDNA insert, was subcloned into M13 and sequenced. Translation of the sequence from this insert demonstrated a single large open reading frame which, upon analysis using the Genbank database, was found to contain a single stretch of about 100 amino acids with sequence homology to SH2 domains of other known proteins (FIGS. 4A to 4I and 5A). However, in other regions, no sequence homology was noted. Thus, using this screening approach, a new SH2-containing protein which could bind to the EGFR was identified.

B. Isolated of Full Length cDNA

The initial clone isolated encoded for an SH2 domain, but did not contain the 3' or 5' ends of the gene. To isolated the full length cDNA, the library was rescreened using DNA isolated from the initial positive phage. DNA, from recombinant M13 bacteriophage which expressed the positive clone, was amplified using a thermal cycler, Taq1 polymerase and oligonucleotides complementary to the EcoR1 flanking regions of the M13 sequence in information, a second amplified DNA product, corresponding to the most 5' 250 nucleotides of the initial isolated phage, was also generated by using oligonucleotides complementary to sequences at both ends of this region. (³² P)-labelled DNA probes were then prepared by nick translation of the amplified products.

To rescreen the cDNA library, the library was replated as described above. After incubation of the plates for 8 hours at 37° C., the plates were cooled for 1 hour at 4° C. following which the phage DNA was transferred to nitrocellulose filters. The filters were denatured in a solution of 0.2N NaOH and 1.5M NaCl and then baked in vacuo for 2 hours at 80° C. (Sambrook, J. et al., (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). After prehybridization of the filters for 1 hour at 42° C., 32P-labelled DNA probe was added and hybridization was continued overnight at 42° C. in a solution containing 5× Denhardt's, 50% formamide, 5× SSC, 0.1% SDS, 200 mM Tris-HCl, pH 7.6 and 100 μg/ml salmon sperm DNA. The filters were then washed in a solution containing 0.1× SSC and 0.1% SDS, dried and exposed to Kodak XAR-5 film at -70° C. Positive clones were then isolated and sequenced as described above.

Since the insert from clone ki4 lacked the 3' and 5' ends of the gene, the library was rescreened using two DNA probes which were generated by amplifying DNA from clone ki4. This approach enabled the identification of five additional clones. Three of the clones extended 3' from the initial clone ki4, two of which, clones, ki2.2 and ki2.4, contained a polyadenylation signal and a long 3' untranslated region (>1000 nucleotides). In addition, these clones encoded a protein which contained a second SH2 domain (FIGS. 4A to 4I and 5A).

The other two clones, ki3.0 and ki5.3, extended 5' from clone ki4. Both clones contained long open reading frees and an AUG codon which met the translation initiation criteria as defined by Kozak (Kozak, M. J. Cell. Biol. 108:229-241 (1989)). However, only clone ki3.0, when translated into protein and compared with known sequences in Genbank, was found to contain a domain of 50 amino acids which was homologous to SH3 domains present in other known proteins. The predicted molecular weight of the full length protein encoded by the overlapping clones, ki2.2 and ki3.0, was about 84 kDa. This new protein was termed GRB-1.

EXAMPLE III GRB-1 Protein Contains SH2 and SH3 Domains

Analysis of the GRB-1 protein sequence by comparison to sequences in the Genbank database revealed the presence of two stretches of about 100 amino acids, starting at amino acids 333 and 624, with sequence homology to SH2 domains of other proteins known to interact with the EGFR (FIG. 5A). While GRB-1 displayed striking homology to other SH2 domains at the protein level, it revealed no significant homology at the DNA level. GRB-1 also contained a segment of about 50 amino acids, located in the N-terminal region, which had sequence homology to SH3 domains (FIGS. 4 and 5B).

A comparison of the structural organization of GRB-1 with several other SH2/SH3 containing proteins is shown in FIG. 6. It is apparent from this scheme that the localization of the SH2 and SH3 domains vary from protein to protein. Despite this there are certain similarities and differences among these SH2 containing proteins. GRB-1 is similar to some other substrates which have been found to interact with the EGFR, such as PLC-δ and GAP, in that GRB-1 contains two SH2 domains and a single SH3 domain. However, unlike these substrates, GRB-1 contains no homology to any known catalytic domain, and in this regard resembles the protein encoded by the avian sarcoma virus, v-crk.

Out side of these regions there was no sequence homology with other protein sequences present in Genbank. In particular, GRB-1 lacked a consensus ATP-binding domain, and did nod display sequence homology with any serine/threonine kinase or tyrosine kinase.

The SH2 domain is thought to provide a common motif by which enzymatically distinct signalling molecules can be coupled to activated receptors with tyrosine kinase activity (Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990); Anderson, D. et al., Science 250:979-982 (1990)).

The presence of SH2 domains in GRB-1 (FIG. 4) and in GRB-2 further reinforces the importance of this domain in mediating the interaction of these proteins with the C-terminal tail of the EGFR. Moreover, since many proteins capable of interacting with cytoplasmic or receptor tyrosine kinases remain to be identified, this suggests that additional members of this protein family remain to be discovered.

In addition to containing two SH2 domains, GRB-1 also contains an SH3 domain. The SH3 domain is a non-catalytic domain of about 50 amino acid residues which is shared among many SH2-containing proteins. Since SH3 domains are also found in cytoskeletal proteins, such as spectrin and fodrin, the function of this domain could be to localize these proteins to the membrane or submembrane cytoskeleton where they would interact with other molecules.

Comparison of the deduced amino acid sequence of GRB-1 with the protein product encoded by the avian oncogene v-crk may shed light on GRB-1 function. The gene v-crk encodes a protein which is composed primarily of a viral gag protein fused to an SH2 and SH3 domain (Mayer, B. J. et al., Nature 332:272-275 (1988)). Both GRB-1 and the p47^(gag-crk) protein have no homology with any known catalytic domains. However, chicken embryo fibroblasts transformed with p47^(gag-crk) display elevated levels of phosphotyrosine-containing proteins (Mayer, B. J. et al., supra; Proc. Natl. Acad. Sci. USA 87:2638-2642 (1990); Matsuda, M. et al., Science 248:1537-1539 (1990)).

Since the v-crk product has been shown to bind several phosphotyrosine-containing proteins in v-crk transformed cells, it may be that the function of c-crk is to act as a bridge between kinases and substrates. In this regard, it is intriguing that GRB-1, like GAP and PLC-δ, contains two SH2 domains, the combination of which may be ideally suited for linking other proteins to activated tyrosine kinase molecules.

EXAMPLE IV Northern Analysis of GRB-1 Expression A. Methods

Total cellular RNA was prepared from monkey tissue by the guanidinium isothiocyanate/cesium chloride method described by Sambrook, J. et al., (supra). Poly (A)+RNA was prepared by oligo(dT) cellulose chromatography. For Northern analysis, RNA was size fractionated by electrophoresis in a 1.2% agarose/2.2M formaldehyde gel, transferred onto a nylon membrane by capillary action and baked at 80° C. for 2 hours. Following prehybridization, the blot was hybridized with a (³² P)-nick-translated DNA probe which was prepared as descried above. Hybridization was carried out overnight at 42° C. in the presence of 50% formamide, 5× SSC, 0.1% SDS, and 5× Denhardt's. The membrane was then washed in 0.1× SSC, 0.1% SDS at 42° C., and exposed to Kodak XAR film at -70° C. for 12 hours using an intensifying screen.

B. Results

To test for the expression of mRNA corresponding to the newly isolated cDNA, Northern blot analysis of different monkey tissue mRNA, probed with DNA corresponding to the insert from clone ki4, demonstrated the presence of two major bands of 4.4 kb and 7.0 kb in most tissues examined (FIG. 7). Expression was highest in the brain, with heart, spleen, liver and thymus displaying decreasing levels of expression. The 4.4 kb message corresponds to the expected size of the transcript which would encode the isolated clones. In contrast to the 4.4 and 7.0 kb transcripts observed in most tissues, the skin contained two slightly smaller sized mRNAs of 3.6 and 6.6 kb.

The 3.6, 6.6 and 7.0 kb transcripts may represent alternatively spliced forms of mRNA, or may encode for distinct but related mRNA species.

EXAMPLE V Production of anti-GRB-1 Antibodies and Analysis of GRB-1 Fusion Protein A. Methods

Polyclonal antibodies were produced by immunizing rabbits with the β-galactosidase fusion protein expressed by the initial isolated phage clone, ki4. E. coli CAG 456 bacteria (obtained from Dr. Michael Snyder, Yale University) were infected with recombinant phage ki4 at a multiplicity-of-infection of 10 and β-galactosidase fusion protein was recovered from the protein pellet after 1.5 hours. Protein extracts were prepared, separated on a 6% SDS-gel, and the band corresponding to the fusion protein excised from gel and used for immunization.

Human glioblastoma cell line U1242, rat bladder carcinoma cell line NBT II, and NIH3T3 cells were grown to confluence in DMEM medium supplemented with 10% fetal bovine serum. Cells were labelled with (³⁵ S)-methionine (50 μCi/ml) in 0.5% fetal bovine serum and lysed after 12 hours as previously described (Margolis, B. et al., Cell 57:1101-1107 (1989)). After immunoprecipitation with 10 μl of antibody coupled to protein A-Sepharose, the beads were washed three times with a solution containing 20 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate. After boiling in sample buffer proteins were separated on a 8% SDS-gel.

B. Results

Polyclonal antibodies were raised against the β-galactosidase fusion protein expressed by the initial isolated phage. Immunoprecipitation experiments, using biosynthetically labelled cells, demonstrated that these antibodies recognized an 85 kDa protein in three different cell lines (FIG. 8, lanes designated "I"). Recognition of the 85 kDa protein by this antiserum was specific since preimmune serum did not recognize this protein (lanes designated "P"). These results provided support for the predicted molecular weight based on the amino acid sequence of cloned GRB-1.

C. Discussion

The finding that the gene for GRB-1 encodes for a protein with an expected molecular weight of 85 kDa, together with the demonstration that antibodies to GRB-1 immunoprecipitated an 85 kDa protein from three different cell lines, suggest that GRB-1 may represent a particular protein which had previously been shown to associate with activated growth factor receptors, namely p85. While the exact function of p85 was unknown, it was presumed to be phosphatidylinositol (PI3)-kinase, since PI3-kinase activity copurified with an 85 kDa protein found in PDGF-stimulated as well as middle T-antigen (MTAg)-transformed cells (Kaplan, D. R. Cell 50:1021-1029 (1987); Whitman, M. et al., Nature 315:239-242 (1985); Coughlin, S. R. et al., Science 243:1191-1194 (1989)). The absence of an ATP binding site argues that GRB-1 is most likely not a phospholipid kinase. GRB-1 exhibits 97% sequence identity with murine and bovine p85. Hence, GRB-1 is the human counterpart of p85. Recombinant p85 is able to bind to the activated PDGFR or EGFR, but does not itself contain intrinsic PI3 kinase activity. p85, however, is found associated with a 110 kDa tyrosine phosphorylated protein which may be the catalytic subunit of the PI3 Kinase. While the exact relationship between PI3 kinase and p85 is not known, overexpression of p85 modulates the interaction between PI3 kinase and the PDGFR. p85 could function as a regulatory subunit or as a bridge between activated receptors and the PI3 kinase.

EXAMPLE VI The Tyrosine Phosphorylated Carboxy-terminus of the EGF Receptor is a Binding Site for GAP and PLC-δ

The studies described below confirm that binding of PLC-δ and a fusion protein containing the SH2 and SH3 domains of GAP (trpE/GAP SH2) are specifically controlled by autophosphorylation of the EGFR. The results show that phosphorylation of PLC-δ actually reduces its association with the EGFR. Evidence is presented demonstrating that both PLC-δ and the trpE/GAP SH2 fusion protein bind specifically to the tyrosine phosphorylated C-terminus of the EGFR. In sum, these results indicate that the SH2/SH3 domains interact directly with phosphotyrosine containing regions of the EGF receptor.

A. Materials and Methods

1. Cell lines, mutant receptors and fusion proteins.

The cell lines CD126 (Margolis, B. L. et al., J. Biol. Chem. 264: 10667-10671 (1989a), HER14, K721 (Honegger, A. M. et al., Cell 51: 199-209 (1987); Honegger, A. M. et al., Mol. Cell. Biol. 7:4567-4571 (1987)) were used as sources for wild-type EGF receptor, kinase-negative (kin⁻) EGF receptor and C-terminal (C-terminal) truncated EGF receptor, respectively. The intracellular domain of the EGF receptor (EGFR-C) was purified from a baculovirus expression system (Hsu, C-.J. et al., Cell Growth Differ 1:191-200 (1990)) (FIG. 9A). 3TP1, a cell line which overexpresses transfected PLC-δ cDNA but has no EGF receptor was used as a source of PLC-δ (Margolis, B. et al., Science 248:607-610 (1990b)).

The preparation of trpE fusion proteins containing the GAP SH2 domain (GAP residues 171-448, FIG. 9B) has been described by Moran, M. F. et al., Proc. Natl Acad. Sci. USA 87: 8622-8626 (1990). Bacterial lysates containing trpE/GAP SH2 fusion proteins were prepared by resuspending 1 g of bacteria in 3 ml of 50 mM Tris pH 7.5, 0.5 mM EDTA, 0.1 mM PMSF. After incubation at 4° C. in 1 mg/ml lysozyme and 0.2% NP-40, cells were sonicated 5 times for 5 seconds, and the lysate was clarified by centrifugation for 30 min at 10,000 g. Bacterial lysates were diluted 1:100 in the 1% Triton lysis buffer with proteinase and phosphatase inhibitors as described above and were precleared with protein A-Sepharose.

2. Antibodies, immunoprecipitation and immunoblotting

The following anti-EGFR antibodies (FIG. 9A) were used: (a) mAb108, a monoclonal antibody directed against domain III of the extracellular domain (Lax, I. et al., EMBO J. 8: 421-427 (1989)); (b) antipeptide antibody RK2 specific for residues 984-996; (c) antipeptide antibody C specific for residues 1176-1186; and (d) antipeptide antibody F, specific for residues 656-676. For immunoprecipitating the trpE fusion proteins, a mouse monoclonal antibody against trpE (Oncogene Science) bound to agarose linked anti-mouse IgG (Sigma) was utilized. For immunoblotting, a polyclonal rabbit antibody against trpE was used (Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990)). PLC-δ was immunoblotted and immunoprecipitated with a polyclonal rabbit anti-peptide antibody described previously (Margolis, B. et al., Cell 57:1101-1107 (1989b)).

The techniques used are described in several references from the present inventors' laboratory (Margolis, B. L. et al., J. Biol. Chem. 264:10667-10671 (1989); Cell 57:1101-1107 (1989)). Unstimulated cells were grown to confluence in Dulbecco's Modified Eagle Medium with 10% calf serum and starved overnight in 1% fetal calf serum prior to lysis in a 1% Triton X-100 lysis buffer containing proteinase and phosphatase inhibitors. EGF receptors were immunoprecipitated utilizing antibodies bound to protein A-Sepharose. After washing the receptor material with HNTG (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and 10% glycerol), autophosphorylation was induced by the addition of 5 mM MnCl₂ and 30 μM ATP. Controls were incubated with Mn²⁺ only. After further washes with HNTG, lysate containing either PLC-δ (from 3TP1 cells) or the bacterial fusion proteins was added. After allowing binding to proceed for 90 min, three further washes with HNTG were performed and samples were run on an SDS gel and immunoblotted.

3. Cyanogen bromide (CNBr) Cleavage

EGFR-C was phosphorylated at 4° C. with MnCl₂ and ATP sometimes in the presence of (δ-³² P)ATP (NEN/Dupont, 6000 Ci/mmol). The receptor preparation was then resuspended in 20 mM HEPES, pH 7.5, with 100 μg BSA and concentrated in a Centricon 10 (Amicon) to 50 μl. Then 240 μl 88% formic acid was added with two grains of CNBr and the samples were stored under nitrogen in the dark for 14 h at room temperature. Samples were dried and washed three times with water in a Speed-Vac (Savant) and then resuspended in 1% Triton lysis buffer.

B. RESETS

A comparison was performed of the binding of PLC-δ to wild-type and mutant EGFRs (FIG. 9A). First, wild-type and mutant receptors from transfected NIH-3T3 cells were immunoprecipitated and some of the receptor immunoprecipitates were allowed to undergo in vitro autophosphorylation with ATP and Mn²⁺ (Margolis, B. et al., Mol. Cell. Biol. 10:435-441 (1990a)). Then, lysates from NIH-3T3 cells which overexpress PLC-δ (Margolis, B. et al., Science 248:607-610 (1990b)) were added and binding allowed to proceed for 90 min. at 4° C. After washing the immunoprecipitates with HNTG, the amount of PLC-δ bound was assessed by immunoblotting. As illustrated in FIGS. 10A-10B, PLC-δ bound only to the tyrosine phosphorylated wild-type receptor but not to the non-phosphorylated receptor.

To assess the importance of autophosphorylation, two studies with mutant receptors were then undertaken. First to be examined was the binding of PLC-δ to a truncated EGF receptor missing 126 amino acids from the C-terminus (CD126, FIG. 9A) and devoid of four major autophosphorylation sites (Downward, J. et al., Nature 311: 483-485 (1984)). This truncated receptor was autophosphorylated, probably at tyrosine 992 (Walton, G. M. et al., J. Biol. Chem. 265: 1750-1754 (1990)). However, despite this level of tyrosine autophosphorylation, the binding of PLC-δ was markedly reduced compared to the full length receptor. Reduced association was also observed with CD63, a deletion mutant EGF receptor lacking 63 C-terminal residues containing two autophosphorylation sites. These results suggested a role for the receptor C-terminus in either binding or modulating the binding of PLC-δ to the EGF receptor.

FIG. 10A-10B also demonstrates that PLC-δ cannot bind to the kin⁻ mutant receptor. To explore the importance of autophosphorylation in this effect, the kin receptor was cross-phosphorylated with the CD126 receptor (Honegger, A. M. et al., Proc. Natl. Acad. Sci. USA 86:925-929 (1989)). This resulted in normalization of PLC-δ binding to wild-type levels. This suggested that phosphorylation of the kin receptor was sufficient to normalize binding to PLC-δ.

To confirm that the kin receptor alone could bind PLC-δ after phosphorylation, this receptor was cross-phosphorylated with a soluble, baculovirus-expressed EGFR cytoplasmic domain (EGFR-C) that does not bind to the mAb 108 (FIG. 9A).

Although cross-phosphorylation was not as strong as with the CD126 mutant, tyrosine phosphorylation of the K721A mutant and binding of PLC-δ were clearly detected. This finding confirms that tyrosine phosphorylation of the EGFR promotes binding of PLC-δ.

The role of PLC-δ tyrosine phosphorylation in the interaction between wild-type EGFR and PLC-δ was examined. Tyrosine phosphorylated PLC-δ could be dissociated from the EGFR more readily than non-phosphorylated PLC-δ (FIG. 11), suggesting a lower affinity of tyrosine phosphorylated PLC-δ for the EGFR.

These findings were extended to examination of the binding of a fusion protein containing trpE/GAP SH2 domain (FIG. 9B) to the baculovirus expressed EGFR-C. As with the full length EGFR and PLC-δ, the trpE/GAP SH2 fusion protein domain bound only to the tyrosine phosphorylated EGFR-C (FIG. 12A). The trpE protein alone did not bind to EGFR-C. Similarly, phosphorylated EGFR-C bound only to trpE/GAP SH2; however, non-specific binding of non-phosphorylated EGFR-C was high (FIG. 12B). These results demonstrated that the binding site of the EGFR is situated in its intracellular domain.

In general, the trpE/GAP SH2 fusion protein bound with a higher stoichiometry to full length EGFR than did PLC-δ. However, the fusion protein was not tyrosine phosphorylated by the EGFR. The trpE/GAP SH2 protein much better to the phosphorylated full length receptor compared to the CD126 deletion mutant (FIG. 13A). As shown in FIG. 13B, cross-phosphorylation of the kin⁻ full length EGF receptor by the EGFR-C allowed it to bind the trpE/GAP SH2 protein.

In control groups, the EGFR-C was shown not to enhance the binding to the CD126 receptor probably because this receptor was already maximally tyrosine phosphorylated (FIG. 13A). Also, no binding was observed when EGFR-C was tested in the presence of mAb 108 immunoprecipitate from cells containing no EGF receptor (FIG. 13B). This indicates that the effects of EGFR-C could not be attributed to non-specific binding of tyrosine phosphorylated EGFR-C to sepharose. These studies confirm the importance of autophosphorylation in mediating binding and show that for EGF receptor binding, the GAP SH2 domain behaves similarly to intact PLC-δ.

The poor binding to the CD126 deletion mutant suggested that at least part of the binding site for the molecule was in the C-terminus. Yet an effect, possibly allosteric, of this deletion on the overall conformation of the receptor could not be excluded. Therefore, the binding of PLC-δ and trpE/GAP SH2 to a C-terminal fragment of the EGFR was examined. In the EGFR, the most C-terminal methionine residue is found at position 983; CNBr cleavage therefore generates a 203 amino acid fragment which contains all the known autophosphorylation sites. This protein fragment is recognized by an antibody specific for the EGFR C-terminus, anti-C (FIG. 9A).

When this C-terminal fragment was specifically immunoprecipitated and tyrosine phosphorylated, it bound PLC-δ and the trpE/GAP SH2 fusion protein (FIG. 14). CNBr cleavage was complete; no full-length EGFR-C could be detected after proteolysis that could account for the binding. Again, no binding was seen to the non-phosphorylated C-terminal CNBr fragment. CNBr cleavage of EGFR-C also generated a 97 amino acid N-terminal peptide identified by antibody F (FIG. 9A, EGFR residues 645-742). This fragment, immunoprecipitated by antibody F, did not bind trpE/GAP SH2. Additionally, EGFR-C was autophosphorylated with (δ-³² P)ATP and a ³² P-labeled CNBr C-terminal fragment was generated. As shown in FIG. 15, this fragment bound to the trpE/GAP SH2 fusion protein but not to trpE. In total, these findings demonstrate that direct binding to the tyrosine phosphorylated C-terminus contributes at least in part to the specific binding of SH2 and SH3 domain proteins to the EGFR.

C. Discussion

When taken together, the above findings and several additional lines of evidence argue strongly that the phosphotyrosine residues are part of the actual binding site of the EGFR for SH2 domains. First, P47^(gag-crk) was found to bind to nearly all phosphotyrosine-containing proteins in v-crk transformed cells (Matsuda, M. et al., Science 248:1537-1539 (1990)). Second, mutations of two autophosphorylation sites on the PDGF receptor greatly decreased the binding of GAP (Kazlauskas, A. et al., Science 247:1578-1581 (1990)). Finally, the results presented above demonstrate specific binding to the C-terminus of the EGFR only when phosphotyrosine is present. Thus, it is concluded that the phosphotyrosine residues either comprise a part of the binding site or locally alter the conformation of this region, allowing binding. It is unlikely that phosphotyrosine alone constitutes the binding site. For example, phosphotyrosine alone cannot interfere with the binding of P47^(gag-crk) to phosphotyrosine-containing proteins (Matsuda et al., supra). Additionally, PLC-δ does not bind to activated all molecules that contain phosphotyrosine residues, such as the CSF-1 receptor (Downing, J. R. et al., EMBO J. 8:3345-3350 (1989)). Similarly, the binding of PLC-δ to PDGFR does not appear to be identical to GAP binding; different SH2 and SH3 domain-containing proteins may have different binding specificities (Kazlauskas et al., supra).

EXAMPLE VII Cloning, Isolation & Characterization of a Target Protein for Receptor Tyrosine Kinase

METHODS: The intracellular domain of the EGFR, which includes the tyrosine kinase and carboxy terminal domain, was purified from a recombinant baculovirus expression system as described (Margolis Mol. Cell. Biol. 10:435-441 (1990) and EMBO J. 9:4375-4390 (1990); Skolnik et al. Cell 65:83-90 (1991). The recombinant protein was phosphorylated with (³² P) δ-ATP, washed, and cyanogen bromide digested to yield a 204 residue carboxyterminal tail containing all five phosphorylated tyrosine residues (Margolis Mol. Cell. Biol. 10:435-441 (1990a) and EMBO J. 9:4375-4390 (1990b). The (³² P)-carboxyterminal tail was then used as probe to screen a λgt11 human brainstem expression library, as previously described (Skolnik et al. Cell 65:83-90 (1991)).

An oligo (dT) λgt11, constructed from mRNA isolated from human brain stem, was obtained from M. Jaye (Rhone Poulenic-Rorer Pharmaceuticals) and is readily available from commercial sources. Screening of the library was performed as previously described (Skolnik et al. Cell 65:83-90 (1991)). cDNA inserts isolated from positive recombinant phage that bound the EGFR were subcloned into M13 and sequenced by the dideoxy chain termination method, using the Sequenase 2.0 kit (U.S.B). Since the initial clone isolated by expression/cloning did not contain the 5' ends of the gene, the library was rescreened, using the clone 2-4 insert as a DNA probe.

Total cellular RNA was prepared with the Stratagene RNA isolation kit. For Northern analysis, RNA was size fractionated on a 1.2% agarose-2.2M formaldehyde gel, transferred by capillary action to a Nytran membrane (Schleicher and Schuell), and prehybridized and hybridized at 65° in 0.5M sodium phosphate pH 7.2, 7% SDS, 1 mM EDTA, 100 ug/ml salmon sperm DNA. The membrane was then washed 1× at room temp and then 2× at 65° C. in 40 mM sodium phosphate pH 7.2, 1% SDS, 1 mM EDTA.

HER14 are NIH 3T3 cells (clone 2.2) which express approximately 400,000 wild type human EGF receptors per cell (Honeggar et al. Cell 51:199-209 (1987)). HER14 cells were maintained in Dulbecco's modified Eagles medium (DMEM) containing 10% calf serum (CS). Prior to stimulation, cells were cultured for 18 hours in DMEM/1% CS. Cells were then stimulated with either EGF (275 ng/ml) or PDGF-BB (50 ng/ml) Intergen, Purchase, N.Y.) for 2 minutes in DMEM containing 1 mg/ml BSA and 20 mM HEPES pH 7.5, following which the cells were immediately washed and lysed. Lysate protein content was normalized as described (Bradford, 1976). Cell lysis, immunoprecipitation, and immunoblotting were performed as previously described (Margolis et al. Cell 57:1101-1107 (1989)). 293 cells were transfected using a modification of the calcium phosphate precipitation method (Chen and (Okayama Mol. Cell. Biol. 7:2745-272 (1987).

Several polyclonal antibodies were generated against GRB2. A synthetic peptide derived from the N-terminal SH3 domain (residues 36-50) and the full length GRB2-GST (glutathione-S-transferase) fusion protein were used to produce rabbit polyclonal antisera called Ab 86 and Ab 55, respectively. Both of these antisera are effective at recognizing denatured GRB2 in immunoblots. A third polyclonal rabbit antisera called Ab50 was generated against the GRB2-GST fusion protein containing the C-terminal SH3 domain of GRB2 (residues 167-221), and is capable of immunoprecipitating GRB2 from solubilized cells. Monoclonal antiphosphotyrosine antibodies (1G2) covalently coupled to agarose were purchased from Oncogene Science (Manhasset, N.Y.). Anti-P-Tyr immunoblots were performed with a rabbit polyclonal antibody. Anti-EGF receptor immunoprecipitates were performed with monoclonal antibody mAb m108 (Bellot et al. J. Cell Biol. 110:491-502 (1990).

Anti-EGF receptor immunoblots were performed with anti-C terminus peptide (residues 1176-1186) antisera (Margolis et al. Cell 57:1101-1107 (1989)).

Using the cDNA of GRB2 as a template, DNA fragments corresponding to the various GRB2 domains were synthesized using PCR and oligonucleotides which contained appropriate restriction sites and bordered the domains of interest. The amplified DNA was isolated, digested with BamHI and EcoRI and cloned into pGEX3X (Pharmacia), which was then used to transform E. coli HB 101 to ampicillin resistance. Large scale cultures were then grown, induced with IPTG, and the glutathione S-transferase (GST) fusion proteins purified on glutathione agarose beads as previously described (Smith and Johnson Gene 67:31-40 (1988)).

The following fusion proteins were prepared: GST-GRB2 full length (FL) (amino acids {AA} 2-217); GST-SH2 (AA 50-161); GST-N-terminal SH3 (AA 2-59); GST-C-terminal SH3 (AA 156-217); GST-N-terminal SH3-SH2 (AA-161); GST-SH22-C-terminal SH3 (AA 50-217).

To assay the binding of native growth factor receptors to GST-fusion proteins 500 ul of HER14 cell lysate was incubated for 90 min at 4° C. with approximately 5 ug of fusion protein coupled to glutathione agarose beads. The beads were then washed three times with HNTG, and after boiling in sample buffer, the proteins were separated on 8% SDS-PAGE. Bound proteins were transferred to nitrocellulose and blotted with antibodies as described (Margolis et al. Mol. Cell. Biol. 10:435-411 (1990a), Margolis et al. EMBO J. 9:4375-4380 (1990B); Margolis Cell Growth and Differentiation 3:73-80 (1992); and Margolis et al. Nature 356:71-74 (1992).

Labeling cells with (³² P)-orthophosphate were carried out as previously described (Li et al. Mol. Biol. Cell 2:641-649, 1991). Briefly, confluent HER14 cells starved for 16 hrs in 1% FCS/DMEM were incubated for two hours in P_(i) -free media, and labeled for two hours in P_(i) -free media, 1% dialyzed FBS, 1 mCi/ml orthophosphate (carrier free, 314.5-337.5 TBq/mmole, purchased form NEN, Wilmington, Del.), at 37° C. Where appropriate, cells were incubated with vanadate (200 uM) at 37° C. for the last 20 minutes of cell labeling. Cells were then stimulated for two minutes with EGF (250 ng/ml) or PDGF (50 ng/ml), rapidly washed 2 times with ice cold phosphate-buffered saline (PBS), and solubilized immediately in lysis buffer (10 mM Tris-Cl pH 7.6, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 uM sodium orthovanadate, 5 uM ZnCl₂, 1 mM PMSF and 0.5% Triton-X-100). After nuclei were removed by centrifugation, the lysates where precleared for 1 hour with 50 ul Sepharose G25, and then incubated overnight with anti-GRB2 antiserum (Ab50) at 4° C. The immune complexes were then precipitated with protein A-Sepharose for 45 min at 4° C., washed 8-15 times with RIPA buffer (20 mM Tris-Cl pH 7.6, 300 mM NaCl, 2 mM EDTA, 1% Triton-X-100, 1% sodium deoxycholate and 0.1% SDS), heated in Laemmli sample buffer containing 0.1M B-mercaptoethanol and 1% SDS at 95° C. for 5 min, resolved by SDS PAGE (8-15% gradient), and visualized by autoradiography of dried gels. To isolate tyrosine phosphorylated proteins, the cell lysates were incubated with anti-PY antibody (Oncogene Science) beads for 2 hours at 4° C. The anti-PY beads were washed 5 times with lysis buffer, followed by elution with phenylphosphate (2 mM) in the presence of ovalbumin.

RESULTS: Isolation of a cDNA clone encoding a protein with novel SH2 and SH3 domains.

The carboxyterminal tail of the EGFR was used as a probe to screen a human brain stem λgt11 protein expression library as previously described (Skolnik et al. Cell 6:4396-4408, 1991). One of the clones isolated utilizing this technique, clone 2-4, contained an insert of 1100 nucleotides found to contain a reading frame encoding novel SH2 and SH3 domains. The insert from clone 2-4 contained a 3' stop codon followed by a polyadenylation signal, but did not contain the 5' start site. To isolate the 5' end of the gene, the library was rescreened using DNA probes generated by amplifying DNA from clone 2-4. This approach enabled identification of clone 10-53, which was found to encode the full length protein. Clone 10-53, while overlapping with clone 2-4 at the 3' end contained a 5' ATG codon meeting Kozak translation initiation criteria (Kozak J. Cell. Biol. 108:229-241 (1989)), giving a 660 bp open reading frame from the initiating methionine (Ficket et el. Nucleic Acids Research 10:5303-5318 (1982)) (FIGS. 26A-26C). Analysis of the protein sequence of clone 10-53 using Genbank revealed that the full length protein contained a single SH2 domain flanked by two SH3 domains, and that these three domains comprise the bulk of the protein (FIG. 26D). The SH2 and SH3 domains of GRB2 are compared to those in other proteins in FIGS. 26C and 26D. The full length protein encoded by clone 10-53 was named GRB2 (for the second growth factor receptor binding protein identified by the CORT method), and encoded a protein with a predicted molecular weight of about 24.5 kDa. The sequence also contains two potential protein kinase C phosphorylation sites (aa 22 and 102), two potential casein kinase 2 phosphorylation consensus sequences (aa 16 and 131) (Woodget et al. Eur. J. Biochem. 161:177-184 1986; Kishimoto et al. J. Biol. Chem. 260:12492-12499 1985; Marin et al. Eur. J. Biochem. 160:239-244 1986; Kuenzel et al. J. Biol. Chem. 262:9136-9140 1987) and two RGD motifs.

Northern Analysis and Protein Expression

To determine tissue distribution of GRB2, Northern hybridization analysis of various mouse tissue RNAs was performed, using as a probe the insert from clone 10-53. This analysis demonstrated GRB2 expression in every tissue examined, with the highest expression in the brain, spleen, lung, and intestine (FIG. 27A). GRB2 transcripts were visible in the thymus upon longer exposure. We have thus far been unable to identify a tissue or cell line which does not express GRB2, further demonstrating the ubiquitous nature of GRB2 expression. GRB2 hybridized to two transcripts of 1.5 and 3.8 kb. The 1.5 kb transcript corresponds to the expected size of clone 10-53.

Several polyclonal rabbit antisera against GRB2 were generated (see methods section) and used to analyze the GRB2 protein by immunoblotting or immunoprecipitation experiments. FIG. 27B shows that a protein of 25 kDa is recognized by the immune, but not by the preimmune antiserum utilizing either immunoprecipitation analysis of (³⁵ S) methionine labelled cells or an immunoblotting approach. The various antisera recognized a 25 kDa protein in every cell line and tissue examined, consistent with the distribution of the GRB2 transcript found in Northern analysis.

GRB2 associates with growth factor receptors in living cells. Receptor substrates which contain SH2 domains are endowed with the ability to physically associate with certain activated growth factor receptors. Since the goal of the CORT cloning technique is to identify target proteins for particular growth factor receptors, we assessed whether GRB2 associates with the EGF receptor. HER 14 cells were treated with or without EGF, lysed, and subjected to immunoprecipitation analysis, according to published procedures (Margolis et al. 1990b, 1991b).

Immunoblotting of anti-EGFR immunoprecipitates with antibodies to GRB-2 demonstrated association of the 25 kDa (GRB-2 protein with activated EGFR (FIG. 28, lane 6). As shown for PLCδ, the association between EGFR and GRB2 was strictly dependent upon ligand activation and tyrosine autophosphorylation (FIG. 28, lanes 5 and 6) (Anderson et al. Science 250:979-982 (1990); Margolis et al. Cell 57:1101-1107 1989, Mol. Cell. Biol. 10:435-441 1990a, EMBO J. 9:4375-4380 1990b; Wahl et al. Natl. Acad. Sci. USA 86:1568-1572 1989, Meisenhelder Cell 57:1109-1122 1989). Thus, GRB2 associates only with the activated tyrosine phosphorylated EGFR. GRB2 was also demonstrated to have an association with EGFR by immunoprecipitation of GRB2 followed by immunoblotting with anti EGF-receptor antibodies (data not shown). Similar results were obtained with PDGF receptor; activated PDGF receptor associated with GRB2 in HER14 cell sin growth factor dependent manner.

However, no association between GRB2 and the FGF receptor was detected when similar experiments, using anti GRB2 for immunoprecipitation and anti FGF receptor antibodies for immunoblotting, were performed with cell lines expressing FGF-receptor (Mohammadi et al. Mol. Cell. Biol. 11:5068-5078 1991).

Interaction of GRB2 with growth factor receptors is mediated via the SH2 domain. It has been shown that SH2 domains mediate the interaction of signalling molecules, such as PLCδ or GAP, with tyrosine phosphorylated growth factor receptors (Koch et al. Science 252:668-674 (1991); Heldin et al. Trends in Biol. Sci. 16:450-452 (1991); Margolis et al. Cell Growth and Differentiation 3:73-80 (1992), Margolis et al. Nature 3556:71-74 1992). In order to determine whether the interaction between GRB2 and growth factor receptors is mediated via the SH₂ domain of GRB2, we constructed bacterial expression vectors which were designed to express GRB2 as well as the various domains of GRB2 as GST-fusion protein (FIG. 4). These fusion proteins were purified by affinity chromatography on glutathione agarose beads (Smith et al. Gene 67:31-40 1988), and subsequently incubated with lysates from EGF- or PDGF-treated HER 14 cells. The ability of the fusion proteins to bind the activated EGF or PDGF receptors was assessed by immunoblotting the washed complexes with either antiphosphotyrosine or anti-receptor antibodies.

Both the full length GRB2 fusion protein and a fusion protein containing only the SH2 domain of GRB2 were each capable of binding tyrosine phosphorylated proteins which comigrated with the activated EGF or PDGF receptors (FIG. 30, lanes 4, 6, 12 and 14). In contrast, neither receptor bound GST alone (FIG. 30, lane 2) nor a GST-fusion protein containing either the amino or carboxy terminal SH3 domains could bind to activated receptors. Binding was ligand dependent, since immunoblotting with anti-EGFR antibodies revealed association of the EGFR with the fusion proteins only when incubated with lysates from growth factor stimulated cells (FIG. 30, lanes 7 through 10). Thus, in agreement with data about other SH2 domain containing proteins, the association between GRB2 and growth factor receptors is mediated by the SH2 domain (Koch et al. Science 252:668-674 1991); Heldin et al. Trends in Biol. Sci. 16:450-452 (1991); Margollis et al. Cell Growth and Differentiation 3:73-80 (1992) and Nature 356:71-74 (1992).

It is noteworthy that the full length GRB2 fusion protein bound several other tyrosine phosphorylated proteins in EGF- and PDGF-stimulated cell lysates (FIG. 30, lanes 3, 4, 11 and 12). While these bound proteins failed to interact with the SH2-GST fusion protein (FIG. 30, lane 6) or either SH3 domain of GRB2 expressed independently, they did interact with a fusion protein containing both the N-terminal SH3 and SH2 domains. The ability of SH3 domain of GRB2 to enhance the binding activity of the SH2 domain suggests that the N-terminal SH3 domain is important for binding to various cellular proteins and that binding to these proteins may require the concerted action of both SH2 and SH3 domains. GRB2 binds to activated growth factor receptors without being phosphorylated in living cells.

After demonstrating that GRB2 was able to bind to activated EGF and PDGF receptors, we were next interested in determining if GRB2 was a substrate for receptor tyrosine kinases. We examined the capacity of EGF to stimulate phosphorylation of GRB2 in HER14 labelled with (³² P)-orthophosphate. These cells were treated with EGF, lysed and immunoprecipitated with antibodies to GRB2. While anti-GRB2 antibodies immunoprecipitated GRB2 from (³⁵ S) methionine labeled cell lysates (FIG. 31, lanes 6 and 8), phosphorylated GRB2 was not detected in the anti-GRB2 immunoprecipitates from orthophosphate labelled cells. Despite marked overexposure of this gel, no detectable band corresponding to GRB2 was evident in the orthophosphate labelled immunoprecipitates. In similar experiments, stimulation of HER14 cells with PDGF also did not result in detectable phosphorylation of GRB2. The failure to detect phosphorylated GRB2 was not due to poor stimulation of the cells by EGF, since anti-P-Tyr immunoprecipitation of the (³² P_(i))-labeled lysates demonstrated a marked increase in tyrosine phosphorylation of numerous cellular substrates following EGF stimulation. Similarly anti-phosphotyrosine immunoblotting of GRB2 immunoprecipitated from EGF- or PDGF-stimulated HER14 cell lysates, did not reveal tyrosine phosphorylated GRB2 (data not shown).

To determine if the failure to detect tyrosine phosphorylated GRB2 was due to the rapid dephosphorylation by a protein tyrosine phosphatase, a potent tyrosine phosphatase inhibitor, vanadate, was tested for its effects upon GRB2 phosphorylation. (³² P)-orthophosphate-labelled cells were incubated with or without vanadate at 37° C. for 20 min prior to the addition of EGF, and GRB2 phosphorylation was assessed as described above. Vanadate treatment of EGF stimulated cells similarly did not result in detectable GRB2 phosphorylation.

The inability to demonstrate GRB2 phosphorylation was further corroborated in a double immunoprecipitation experiment. (³² P)-labeled HER 14 lysates were immunoprecipitated with anti-PTyr antibodies bound to beads, eluted and the eluates subjected to a second immunoprecipitation with anti-GRB2 antibodies. While clear stimulation of tyrosine phosphorylation was demonstrated in these lysates no significant phosphorylation of the antiP-Tyr-associated GRB2 fraction was detected. Thus, our data demonstrates that while GRB2 associates with the EGF and PDGF-receptors it is not a good substrates for either receptors, and that GRB2 is not phosphorylated by a tyrosine or serine/threonine kinase acting later in the signaling pathway induced by ligand binding. This data suggests that growth factor regulation of GRB2 is not mediated through GRB2 phosphorylation.

GRB2 tyrosine phosphorylation was detected in 293 cells transiently overexpressing PDGFR and GRB2 as determined by anti-PTyr and anti-GRB2 blotting (data not shown). A shift in the mobility of GRB2 was detected on anti-GRB2 (Ab86) blots, in the presence of activated PDGF receptor and the lower mobility form was shown to be tyrosine phosphorylated by anti-PTyr blotting. Similar experiments have confirmed that the immunoprecipitating antibody (Ab50) will recognize tyrosine phosphorylated GRB2. This data suggest that it is possible to tyrosine phosphorylate GRB2 under conditions of overexpression of both receptor and GRB2 protein.

Interestingly, a phosphoprotein of approximately 55 kDa was found to con-immunoprecipitate with GRB2 using immune, but not preimmune sera, in lysates from EGF or PDGF stimulated HER14 cells (FIG. 31, lanes 3, 4 and 7, 8). The association of the 55 kDa protein with GRB2 immunoprecipitates was dependent upon growth factor stimulation, since this interaction was not observed in GRB2 immunoprecipitates from unstimulated cell lysates. The identity of this protein is unknown. GRB2 represents the human homologue of the C. elegans gene product sem-5

As mentioned earlier, GRB2 is composed of one SH2 domain flanked by two SH3 domains in the order of SH3, SH2, SH3. A C. elegans gene encoding for a protein with similar size and domain order has been cloned in the laboratory of R. Horvitz (Clark et al., 1992). This gene, called sem-5, plays a crucial role in C. elegans development as mutations in sem-5 impair both vulval development and sex myoblast migration. FIG. 32 shows a comparison of the amino acid sequences of GRB2 and sem-5. The N-SH3 domains are 58% (63%) and the C-terminal SH3 domains are 58% identical (60%), respectively. The overall sequence identity (similarity) is 58% (63%). Considering the evolutionary distance between human and nematode, these two genes are very similar suggesting the sem-5 represents the C. elegans homologue of GRB2.

DISCUSSION

A novel EGF receptor binding protein of the present invention was cloned by the CORT expression cloning method of the present invention, designated as GRB2. This 25 kDa protein contains on SH2 domain and two SH3 domains. GRB2 is widely expressed, as determined by Northern analysis in ten different murine tissues. It is also expressed in every human, monkey and murine cell line tested as revealed by Northern blotting, immunoprecipitation and immunoblotting experiments. Also shown is that GRB2 associates with EGF and PDGF receptors in a ligand-dependent manner, both in vitro and in living cells. Like other SH2-domain containing proteins, the association between GRB2 and growth factor receptors is mediated by the SH2 domain, can be dependent upon receptor tyrosine autophosphorylation, and involves a direct interaction between GRB2 and the tyrosine phosphorylated receptors.

Despite the fact that GRB2 forms stable complexes with tyrosine phosphorylated, on tyrosine, serine, or threonine residues at physiologic levels of expression to any significant extent. The fact that pretreatment of cells with vanadate did not increase GRB2 phosphorylation indicates that GRB2 is not rapidly dephosphorylated by tyrosine phosphatases.

The extent of sequence homology between GRB2 and sem-5 is striking considering the evolutionary distance between nematode and man. The 58% sequence identity (63% similarity) and the conserved overall architecture of these tow proteins suggest that sem-5 and C. elegans homologue of GRB2 or a closely related member of the same gene family. The similarity between GRB2 and sem-5 is higher than the similarity between let-23 and EGFR; approximately 44% and 28.7% sequence similarities in the catalytic kinase and ligand binding domain, respectively (Aroian et al. Nature 348:693-699 1990).

By detailed genetic studies the laboratories of Horvitz and Sternberg have identified gene crucial for C. elegans vulval development and sex myoblast migration (Horvitz and Sternberg Nature 351:535-341 1991; Aroian et al. Nature 348693-699 1990; Clark et al. Nature In press 1992). It was shown that mutation sin let-23 (EGFR like), sem-5 (GRB2) or let-60 (ras like) lead to defects in vulval development, while sem-5 also functions in sex myoblast migration. It was therefore proposed that the products of these genes lie along the same signal transduction pathway crucial for normal vulval development. Hence, on the basis of genetic studies of C. elegans (Horvitz and Sternberg Nature 351:535-541; Aroian et al. Nature 348:693-699; Clark Nature in press 1992), previous studies on growth factor receptors (Ullrich and Schlessinger Cell 61:203-211 (1990)) and the results presented in this report it is possible to propose a model for the information flow and interaction among these proteins in C. elegans and mammalian cells (FIG. 33). Because of the similarity of sem-5 with GRB2 and let-23 with the EGFR it is likely that sem-5 with GRB2 and let-23 with the EGFR it is likely that sem-5 will bind tyrosine phosphorylated let-23 via its SH2 domain according to the scheme presented in FIG. 8. Since mutations in let-60 cause a similar phenotype as mutations in either let-23 and Sem-5, and since activated ras can rescue let-23 and sem-5 mutations, it is reasonable to assume the let-60/ras functions downstream from EGFR and GRB2 and that GRB2 is somehow involved in regulation of ras activity. In this regard, the 55 kDa phosphoprotein which binds to GRB2 in response to growth factor stimulation is expected to be a downstream signaling molecule regulated upon GRB2 binding to activated growth factor receptors.

EXAMPLE VIII Utilization of an Alternative Phage Library Expression System for Detecting Proteins of the Present Invention

A T7 phage library expression system, used an alternative to the phage λgt11 system described in Example II above, was used to express tyrosine kinase target proteins, as presented in the above Examples, with modifications as described below. A T7 polymerase system (Palazzalo et al., Gene 88, 25 (1990); λEXlox vector, Novagen, Inc.), based on the PET expression systems of Studier and coworkers (Studier et al Meth. Enzymol. 185:60 (1990)) fusing cDNA clones to a fragment of the T7 capsid protein T10 under the control of the T7 promoter. These phages were then used to infect E. coli harboring the T7 polymerase under lacUV5 control. Induction with IPTG generated the T7 polymerase which then initiated transcription of the fusion protein encoded by the phage library. The SH2 domain fragment of PLC-δ1 was incorporated into this phage and analyzed the binding of the phosphorylated EGFR, as described in the above Examples. The DNA fragment containing the human PLC-δ1 (Burgess et al., Mol. Cell. Biol. 10, 4770 (1990)) was amplified by PCT with primers that incorporated EcoR1 sites such that the PLC-δ1 fragment would be in the correct reading frame for λgt11. The amplified DNA was cut with EcoR1 and ligated into EcoR1 digested λgt11 DNA (Promega). After packaging (Gigapack, Stragene), the phages were plated and screened with PLC-δ1 antibody using known techniques (Huynh, T. V. et al. In: DNA CLONING, ed. Glover, IRL Press, Oxford, 1:49-78 (1985)). This phage was then tested for binding to a cyanogen bromide generated fragment from ³² P-ATP labelled EGFR as described in the above Examples. An identical approach was taken to clone the two SH2 domains into λgt11 or λEXlox vectors.

As can be seen in FIGS. 25A-C, uniform binding of the EGFR was seen in the that appeared stronger than was seen with the λgt11 system (compare FIG. 25A and 25B). We also cloned in a longer fragment which ran from 532-1290 of PLCδ1 and this was also easily seen in the T7 system (FIG. 25C). The T7 plaques although mostly smaller than the λgt11 plaques gave stronger signals. This makes this system particularly suitable for library screening when there as thousands of small plaques per plate. The major advantage of this system is the high level of protein expression due to the greater activity of the T7 polymerase versus E. coli RNA polymerase. It may also be that the fusion proteins using the smaller T10 gene fragment (26 kd versus the 110 kd B-galactosidase of λgt11) yields more stable expression and that its hydrophobic character promotes binding to nitrocellulose. In addition to directional cloning, the λEXlox phages also allow for automatic conversion to a PET plasmid (Palazzalo et al., Gene 88, 25 (1990)) which can be useful for expression of a fusion protein for antibody production. Accordingly, screening an T7 expression library is expected to give superior results than for λgt11 for such a cloning strategy of the present invention.

Of 1.6 million clones of a directional oligo dT primed mouse T7 (λEXlox) library screened, nine positive clones were obtained. The library from a 16 day mouse embryo was obtained from Novagen. The library was plated at 40,000 phages per plate in E. coli pLysS according to known methods. After growth for 8 hours, plates were covered with nitrocellulose impregnated with 1 mM IPTG. Plates were grown overnight and the filters probed as described in the above Examples. Positive clones were selected and reprobed until plaques were purified. Phages were then converted to plasmids utilizing the bacterial strain Bm25.5 per manufacturer's instruction. These plasmids were used to transform bacterial strain DH5∝ and the resultant plasmids subjected to double stranded sequencing using known techniques (Sequenase Version 2, U.S. Biochemical). Six of nine clones encoded proteins that were similar or identical to other known genes which contained SH2 domains TABLE I--see attached. Figures). The comparison of two of these protein sequences of the present invention, GRB-3 and GRB-4, to their known counterparts is displayed in FIG. 17 and 18. Partial sequence of three clones revealed that they were closely related to the avian oncogene v-crk. GRB-3 has a high degree of identity with v-crk beginning with the methionine at residue 32 and this methionine has been found to be the start site of avian c-crk. In the sequence carboxy-terminus to this methionine, there is 77% amino acid homology (FIG. 17) and 80% DNA similarity between v-crk and GRB-3. GRB-4, was similar to nck (FIG. 18), a human protein composed of three SH3 domains and one SH2 domain. Our clone contained one SH3 domain and one SH2 domain and was 74% identical at the protein level and 66% similar at the DNA level in the open reading frame. We also cloned two SH2 domain proteins with intrinsic enzymatic activity.

                  TABLE I                                                          ______________________________________                                         SH2 DOMAIN   CLONES                                                            PROTEIN      ISOLATED     DESCRIPTION                                          ______________________________________                                         GRB-3        #19,#76,#80  crk-like                                             GRB-4        #64          nck-like                                             GRB-5        #63B         fyn                                                  GRB-6        #88          PLC-γ1                                         GRB-7        #63A,#66,#88 novel protein                                        ______________________________________                                    

A remaining clone encoded a new protein with a unique SH2 domain as GRB-7. To obtain a full length DNA clone, the T7 (λEXlox) library was plated in an E. coli strain without T7 polymerase gene and routine DNA hybridization performed with a 700 base pair EcoR1 fragment from the GRB-7 clone using standard published techniques (Ausubel et al eds., Current Protocols in Molecular Biology, Wiley Interscience, New York, (1987, 1992)). Several overlapping clones were identified which were used for DNA sequencing to obtain the full length GRB-7 protein sequence shown in FIG. 19. A schematic representation of GRB-7 is displayed in FIG. 20 depicting the regions of similarity to known proteins as discussed below. The protein is 535 amino acids in length and has one SH2 domain at its extreme carboxy-terminus. In FIG. 21, the SH2 domain of GRB-7 is compared to other SH2 domains including mouse fyn, human PLC-δ1 and the crk and nck-like proteins we cloned in this project. One interesting aspect is that GRB-7 has an isoleucine at amino acid 448, whereas other SH2 domains have a leucine at this position. To look for other protein motifs in GRB-7, a sequence of 433 amino acids from GRB-7 which excluded the SH2 domain was used to scan the Swissprot and GenEmbl databases, as described herein. Amino acids 242 to 339 of GRB-7, showed similarity to a sequence from the central region of ras GAP. Over this region of 91 amino acids from ras GAP, GRB-7 has 26% identity and 42% similarity allowing for conservative substitutions (FIG. 22). This region of ras GAP lies between the SH2/SH3 domains and he GTPase activating carboxyterminal region and has not been assigned a specific function. The amino-terminal sequence of GRB-7 was found to be proline rich and thus has similarity to many other proline rich proteins. GRB-7 does have an extended region of limited similarity to the catalytic domain of protein phosphatase 2B including this proline rich region (FIG. 23) but no significant similarity was found to other serine/threonine phosphatase such as protein phosphatase 1 or 2A.

A northern blot of GRB-7 in mouse tissues is presented in FIG. 24. Oligo dt selected mRNA was probed with the same EcoR1 fragment used to isolate full length GRB-7. See Ausubel et al eds., Current Protocols in Molecular Biology, Wiley Interscience, New York, (1987, 1992) and Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990). The mRNA was extracted from six week old mice tissues by known methods, e.g., as described by Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990). Approximately 3 μg was run on a 1.2% agarose formaldehyde gel and blotted to nytran (Schleicher and Scheull). The blot was probed with a DNA fragment that encodes amino acids 297 to 515 and labelled with ³² P-dCTP using a random priming labeling kit (U.S. Biochemical). Blots were probed in 0.5M sodium phosphate, pH 7.2, 7% sodium dodecyl sulfate and 1 mM EDTA at 65° C. overnight. Blots were washed in 40 mM sodium phosphate, pH 7.2, 1% SDS and 1 mM EDTA at 65° C. After exposure of the GRB-7 blot for 4 days, blots were stripped and reprobed with actin (exposure 36 hours). The highest signal was detected in liver and kidney, but was also detected in ovary and testes. On longer exposure, a weak signal was detected in lung.

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the generic concept of the present invention. Therefore, such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 50                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3372 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 43..2214                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        TACAACCAGGCTCAACTGTTGCATGGTAGCAGATTTGCAAACATGAGTGCTGAG54                       MetSerAlaGlu                                                                   GGGTACCAGTACAGAGCGCTGTATGATTATAAAAAGGAAAGAGAAGAA102                            GlyTyrGlnTyrArgAlaLeuTyrAspTyrLysLysGluArgGluGlu                               5101520                                                                        GATATTGACTTGCACTTGGGTGACATATTGACTGTGAATAAAGGGTCC150                            AspIleAspLeuHisLeuGlyAspIleLeuThrValAsnLysGlySer                               253035                                                                         TTAGTAGCTCTTGGATTCAGTGATGGACAGGAAGCCAGGCCTCGAAGA198                            LeuValAlaLeuGlyPheSerAspGlyGlnGluAlaArgProArgArg                               404550                                                                         AATGGCTGGTTAAATGGCTATAATGAAACCACAGGGGAAAAGGGGGAC246                            AsnGlyTrpLeuAsnGlyTyrAsnGluThrThrGlyGluLysGlyAsp                               556065                                                                         TTTCCGGGAACTTACGTAGAATATATTGGAAGGAAAAAAATCTCGCCT294                            PheProGlyThrTyrValGluTyrIleGlyArgLysLysIleSerPro                               707580                                                                         CCCACACCAAAGCCCCGGCCACCTCGGCCTCTTCCTGTTGCACCAGGT342                            ProThrProLysProArgProProArgProLeuProValAlaProGly                               859095100                                                                      TCTTCGAAAACTGAAGCAGATGTTGAACAACAAGCTTTGACTCTCCCG390                            SerSerLysThrGluAlaAspValGluGlnGlnAlaLeuThrLeuPro                               105110115                                                                      GATCTTGCAGAGCAGTTTGCCCCTCCTGACATTGCCCCGCCTCTTCTT438                            AspLeuAlaGluGlnPheAlaProProAspIleAlaProProLeuLeu                               120125130                                                                      ATCAAGCTCGTGGAAGCCATTGAAAAGAAAGGTCTGGAATGTTCAACT486                            IleLysLeuValGluAlaIleGluLysLysGlyLeuGluCysSerThr                               135140145                                                                      CTATACAGAACACAGAGCTCCAGCAACCTGGCAGAATTACGACAGCTT534                            LeuTyrArgThrGlnSerSerSerAsnLeuAlaGluLeuArgGlnLeu                               150155160                                                                      CTTGATTGTGATACACCCTCCGTGGACTTGGAAATGATCGATGTGCAC582                            LeuAspCysAspThrProSerValAspLeuGluMetIleAspValHis                               165170175180                                                                   GTTTTGGCTGACGCTTTCAAACGCTATCTCCTGGACTTACCAAATCCT630                            ValLeuAlaAspAlaPheLysArgTyrLeuLeuAspLeuProAsnPro                               185190195                                                                      GTCATTCCAGCAGCCGTTTACAGTGAAATGATTTCTTTAGCTCCAGAA678                            ValIleProAlaAlaValTyrSerGluMetIleSerLeuAlaProGlu                               200205210                                                                      GTACAAAGCTCCGAAGAATATATTCAGCTATTGAAGAAGCTTATTAGG726                            ValGlnSerSerGluGluTyrIleGlnLeuLeuLysLysLeuIleArg                               215220225                                                                      TCGCCTAGCATACCTCATCAGTATTGGCTTACGCTTCAGTATTTGTTA774                            SerProSerIleProHisGlnTyrTrpLeuThrLeuGlnTyrLeuLeu                               230235240                                                                      AAACATTTCTTCAAGCTCTCTCAAACGTCCAGCAAAAATCTGTTGAAT822                            LysHisPhePheLysLeuSerGlnThrSerSerLysAsnLeuLeuAsn                               245250255260                                                                   GCAAGAGTACTCTCTGAAATTTTCAGCCCTATGCTTTTCAGATTCTCA870                            AlaArgValLeuSerGluIlePheSerProMetLeuPheArgPheSer                               265270275                                                                      GCAGCCAGCTCTGATAATACTGAAAACCTCATAAAAGTTATAGAAATT918                            AlaAlaSerSerAspAsnThrGluAsnLeuIleLysValIleGluIle                               280285290                                                                      TTAATCTCAACTGAATGGAATGAACGACAGCCTGCACCAGCACTGCCT966                            LeuIleSerThrGluTrpAsnGluArgGlnProAlaProAlaLeuPro                               295300305                                                                      CCTAAACCACCAAAACCTACTACTGTAGCCAACAACGGTATGAATAAC1014                           ProLysProProLysProThrThrValAlaAsnAsnGlyMetAsnAsn                               310315320                                                                      AATATGTCCTTACAAAATGCTGAATGGTACTGGGGAGATATCTCGAGG1062                           AsnMetSerLeuGlnAsnAlaGluTrpTyrTrpGlyAspIleSerArg                               325330335340                                                                   GAAGAAGTGAATGAAAAACTTCGAGATACAGCAGACGGGACCTTTTTG1110                           GluGluValAsnGluLysLeuArgAspThrAlaAspGlyThrPheLeu                               345350355                                                                      GTACGAGATGCGTCTACTAAAATGCATGGTGATTATACTCTTACACTA1158                           ValArgAspAlaSerThrLysMetHisGlyAspTyrThrLeuThrLeu                               360365370                                                                      AGGAAAGGGGGAAATAACAAATTAATCAAAATATTTCATCGAGATGGG1206                           ArgLysGlyGlyAsnAsnLysLeuIleLysIlePheHisArgAspGly                               375380385                                                                      AAATATGGCTTCTCTGACCCATTAACCTTCAGTTCTGTGGTTGAATTA1254                           LysTyrGlyPheSerAspProLeuThrPheSerSerValValGluLeu                               390395400                                                                      ATAAACCACTACCGGAATGAATCTCTAGCTCAGTATAATCCCAAATTG1302                           IleAsnHisTyrArgAsnGluSerLeuAlaGlnTyrAsnProLysLeu                               405410415420                                                                   GATGTGAAATTACTTTATCCAGTATCCAAATACCAACAGGATCAAGTT1350                           AspValLysLeuLeuTyrProValSerLysTyrGlnGlnAspGlnVal                               425430435                                                                      GTCAAAGAAGATAATATTGAAGCTGTAGGGAAAAAATTACATGAATAT1398                           ValLysGluAspAsnIleGluAlaValGlyLysLysLeuHisGluTyr                               440445450                                                                      AACACTCAGTTTCAAGAAAAAAGTCGAGAATATGATAGATTATATGAA1446                           AsnThrGlnPheGlnGluLysSerArgGluTyrAspArgLeuTyrGlu                               455460465                                                                      GAATATACCCGCACATCCCAGGAAATCCAAATGAAAAGGACAGCTATT1494                           GluTyrThrArgThrSerGlnGluIleGlnMetLysArgThrAlaIle                               470475480                                                                      GAAGCATTTAATGAAACCATAAAAATATTTGAAGAACAGTGCCAGACC1542                           GluAlaPheAsnGluThrIleLysIlePheGluGluGlnCysGlnThr                               485490495500                                                                   CAAGAGCGGTACAGCAAAGAATACATAGAAAAGTTTAAACGTGAAGGC1590                           GlnGluArgTyrSerLysGluTyrIleGluLysPheLysArgGluGly                               505510515                                                                      AATGAGAAAGAAATACAAAGGATTATGCATAATTATGATAAGTTGAAG1638                           AsnGluLysGluIleGlnArgIleMetHisAsnTyrAspLysLeuLys                               520525530                                                                      TCTCGAATCAGTGAAATTATTGACAGTAGAAGAAGATTGGAAGAAGAC1686                           SerArgIleSerGluIleIleAspSerArgArgArgLeuGluGluAsp                               535540545                                                                      TTGAAGAAGCAGGCAGCTGAGTATCGAGAAATTGACAAACGTATGAAC1734                           LeuLysLysGlnAlaAlaGluTyrArgGluIleAspLysArgMetAsn                               550555560                                                                      AGCATTAAACCAGACCTTATCCAGCTGAGAAAGACGAGAGACCAATAC1782                           SerIleLysProAspLeuIleGlnLeuArgLysThrArgAspGlnTyr                               565570575580                                                                   TTGATGTGGTTGACTCAAAAAGGTGTTCGGCAAAAGAAGTTGAACGAG1830                           LeuMetTrpLeuThrGlnLysGlyValArgGlnLysLysLeuAsnGlu                               585590595                                                                      TGGTTGGGCAATGAAAACACTGAAGACCAATATTCACTGGTGGAAGAT1878                           TrpLeuGlyAsnGluAsnThrGluAspGlnTyrSerLeuValGluAsp                               600605610                                                                      GATGAAGATTTGCCCCATCATGATGAGAAGACATGGAATGTTGGAAGC1926                           AspGluAspLeuProHisHisAspGluLysThrTrpAsnValGlySer                               615620625                                                                      AGCAACCGAAACAAAGCTGAAAACCTGTTGCGAGGGAAGCGAGATGGC1974                           SerAsnArgAsnLysAlaGluAsnLeuLeuArgGlyLysArgAspGly                               630635640                                                                      ACTTTTCTTGTCCGGGAGAGCAGTAAACAGGGCTGCTATGCCTGCTCT2022                           ThrPheLeuValArgGluSerSerLysGlnGlyCysTyrAlaCysSer                               645650655660                                                                   GTAGTGGTGGACGGCGAAGTAAAGCATTGTGTCATAAACAAAACAGCA2070                           ValValValAspGlyGluValLysHisCysValIleAsnLysThrAla                               665670675                                                                      ACTGGCTATGGCTTTGCCGAGCCCTATAACTTGTACAGCTCTCTGAAA2118                           ThrGlyTyrGlyPheAlaGluProTyrAsnLeuTyrSerSerLeuLys                               680685690                                                                      GAACTGGTGCTACATTACCAACACACCTCCCTTGTGCAGCACACCGAC2166                           GluLeuValLeuHisTyrGlnHisThrSerLeuValGlnHisThrAsp                               695700705                                                                      TCCCTCAATGTCACACTAGCCTACCCAGTATATGCACAGCAGAGGCGA2214                           SerLeuAsnValThrLeuAlaTyrProValTyrAlaGlnGlnArgArg                               710715720                                                                      TGAAGCGCTTACTCTTTGATCCTTCTCCTGAAGTTCAGCCACCCTGAGGCCTCTGGAAAG2274               CAAAGGGCTCCTCTCCAGTCTGATCTGTGAATTGAGCTGCAGAAACGAAGCCATCTTTCT2334               TTGGATGGGACTAGAGCTTTCTTTGACAAAAAAGAAGTAGGGGAAGACATGCAGCCTAAG2394               GCTGTATGATGACCACACGTTCCTAAGCTGGAGTGCTTATCCCTTCTTTTTCTTTTTTTC2454               TTTGGTTTAATTTAAAGCCACAACCACATACAACACAAAGAGAAAAAGAAATGCAAAAAT2514               CTCTGCGTGCAGGGACAAAGAGGCCTTTAACCATGGTGCTTGTTAATGCTTTCTGAAGCT2574               TTACCAGCTGAAAGTTGGGACTCTGGAGAGCGGAGGAGAGAGAGGCAGAAGAACCCTGGC2634               CTGAGAAGGTTTGGTCCAGCCTGGTTTAGCCTGGATGTTGCTGTGCACGGTGGACCCAGA2694               CACATCGCACTGTGGATTATTTCATTTTGTAACAAATGAACGATATGTAGCAGAAAGGCA2754               CGTCCACTCACAAGGGACGCTTTGGGAGAATGTCAGTTCATGTATGTTCAGAAGAAATTC2814               TGTCATAGAAAGTGCCAGAAAGTGTTTAACTTGTCAAAAAACAAAAACCCAGCAACAGAA2874               AAATGGAGTTTGGAAAACAGGACTTAAAATGACATTCAGTATATAAAATATGTACATAAT2934               ATTGGATGACTAACTATCAAATAGATGGATTTGTATCAATACCAAATAGCTTCTGTTTTG2994               TTTTGCTGAAGGCTAAATTCACAGCGCTATGCAATTCTTAATTTTCATTAAGTTGTTATT3054               TCAGTTTTAAATGTACCTTCAGAATAAGCTTCCCCACCCCAGTTTTTGTTGCTTGAAAAT3114               ATTGTTGTCCCGGATTTTTGTTAATATTCATTTTTGTTATCCTTTTTTAAAAATAAATGT3174               ACAGGATGCCAGTAAAAAAAAAAATGGCTTCAGAATTAAAACTATGAAATATTTTACAGT3234               TTTTCTTGTACAGAGTACTTGCTGTTAGCCCAAGGTTAAAAAGTTCATAACAGATTTTTT3294               TTGGACTGTTTTGTTGGGCAGTGCCTGATAAGCTTCAAAGCTGCTTTATTCAATAAAAAA3354               AAAACCCGAATTCACTGG3372                                                         (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 724 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetSerAlaGluGlyTyrGlnTyrArgAlaLeuTyrAspTyrLysLys                               151015                                                                         GluArgGluGluAspIleAspLeuHisLeuGlyAspIleLeuThrVal                               202530                                                                         AsnLysGlySerLeuValAlaLeuGlyPheSerAspGlyGlnGluAla                               354045                                                                         ArgProArgArgAsnGlyTrpLeuAsnGlyTyrAsnGluThrThrGly                               505560                                                                         GluLysGlyAspPheProGlyThrTyrValGluTyrIleGlyArgLys                               65707580                                                                       LysIleSerProProThrProLysProArgProProArgProLeuPro                               859095                                                                         ValAlaProGlySerSerLysThrGluAlaAspValGluGlnGlnAla                               100105110                                                                      LeuThrLeuProAspLeuAlaGluGlnPheAlaProProAspIleAla                               115120125                                                                      ProProLeuLeuIleLysLeuValGluAlaIleGluLysLysGlyLeu                               130135140                                                                      GluCysSerThrLeuTyrArgThrGlnSerSerSerAsnLeuAlaGlu                               145150155160                                                                   LeuArgGlnLeuLeuAspCysAspThrProSerValAspLeuGluMet                               165170175                                                                      IleAspValHisValLeuAlaAspAlaPheLysArgTyrLeuLeuAsp                               180185190                                                                      LeuProAsnProValIleProAlaAlaValTyrSerGluMetIleSer                               195200205                                                                      LeuAlaProGluValGlnSerSerGluGluTyrIleGlnLeuLeuLys                               210215220                                                                      LysLeuIleArgSerProSerIleProHisGlnTyrTrpLeuThrLeu                               225230235240                                                                   GlnTyrLeuLeuLysHisPhePheLysLeuSerGlnThrSerSerLys                               245250255                                                                      AsnLeuLeuAsnAlaArgValLeuSerGluIlePheSerProMetLeu                               260265270                                                                      PheArgPheSerAlaAlaSerSerAspAsnThrGluAsnLeuIleLys                               275280285                                                                      ValIleGluIleLeuIleSerThrGluTrpAsnGluArgGlnProAla                               290295300                                                                      ProAlaLeuProProLysProProLysProThrThrValAlaAsnAsn                               305310315320                                                                   GlyMetAsnAsnAsnMetSerLeuGlnAsnAlaGluTrpTyrTrpGly                               325330335                                                                      AspIleSerArgGluGluValAsnGluLysLeuArgAspThrAlaAsp                               340345350                                                                      GlyThrPheLeuValArgAspAlaSerThrLysMetHisGlyAspTyr                               355360365                                                                      ThrLeuThrLeuArgLysGlyGlyAsnAsnLysLeuIleLysIlePhe                               370375380                                                                      HisArgAspGlyLysTyrGlyPheSerAspProLeuThrPheSerSer                               385390395400                                                                   ValValGluLeuIleAsnHisTyrArgAsnGluSerLeuAlaGlnTyr                               405410415                                                                      AsnProLysLeuAspValLysLeuLeuTyrProValSerLysTyrGln                               420425430                                                                      GlnAspGlnValValLysGluAspAsnIleGluAlaValGlyLysLys                               435440445                                                                      LeuHisGluTyrAsnThrGlnPheGlnGluLysSerArgGluTyrAsp                               450455460                                                                      ArgLeuTyrGluGluTyrThrArgThrSerGlnGluIleGlnMetLys                               465470475480                                                                   ArgThrAlaIleGluAlaPheAsnGluThrIleLysIlePheGluGlu                               485490495                                                                      GlnCysGlnThrGlnGluArgTyrSerLysGluTyrIleGluLysPhe                               500505510                                                                      LysArgGluGlyAsnGluLysGluIleGlnArgIleMetHisAsnTyr                               515520525                                                                      AspLysLeuLysSerArgIleSerGluIleIleAspSerArgArgArg                               530535540                                                                      LeuGluGluAspLeuLysLysGlnAlaAlaGluTyrArgGluIleAsp                               545550555560                                                                   LysArgMetAsnSerIleLysProAspLeuIleGlnLeuArgLysThr                               565570575                                                                      ArgAspGlnTyrLeuMetTrpLeuThrGlnLysGlyValArgGlnLys                               580585590                                                                      LysLeuAsnGluTrpLeuGlyAsnGluAsnThrGluAspGlnTyrSer                               595600605                                                                      LeuValGluAspAspGluAspLeuProHisHisAspGluLysThrTrp                               610615620                                                                      AsnValGlySerSerAsnArgAsnLysAlaGluAsnLeuLeuArgGly                               625630635640                                                                   LysArgAspGlyThrPheLeuValArgGluSerSerLysGlnGlyCys                               645650655                                                                      TyrAlaCysSerValValValAspGlyGluValLysHisCysValIle                               660665670                                                                      AsnLysThrAlaThrGlyTyrGlyPheAlaGluProTyrAsnLeuTyr                               675680685                                                                      SerSerLeuLysGluLeuValLeuHisTyrGlnHisThrSerLeuVal                               690695700                                                                      GlnHisThrAspSerLeuAsnValThrLeuAlaTyrProValTyrAla                               705710715720                                                                   GlnGlnArgArg                                                                   (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 770 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 3..770                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        AGCCTGACACCGGAGCCGGTCCGCTGGGCGCGGGCGCCAGGGCTGGA47                              ProAspThrGlyAlaGlyProLeuGlyAlaGlyAlaArgAlaGly                                  151015                                                                         GGGGCGCGCGTGCCGGCGGCGGCCCAGCGTGAAAGCGCGGAGGCGGCC95                             GlyAlaArgValProAlaAlaAlaGlnArgGluSerAlaGluAlaAla                               202530                                                                         ATGGCGGGCAACTTCGACTCGGAGGAGCGGAGTAGCTGGTACTGGGGC143                            MetAlaGlyAsnPheAspSerGluGluArgSerSerTrpTyrTrpGly                               354045                                                                         CGCCTGAGCCGGCAGGAGGCGGTGGCGCTATTGCAGGGCCAGCGCGAC191                            ArgLeuSerArgGlnGluAlaValAlaLeuLeuGlnGlyGlnArgAsp                               505560                                                                         GGGGTGTTCCTGGTGCGGGACTCGAGCACCAGCCCCGGGGACTATGTG239                            GlyValPheLeuValArgAspSerSerThrSerProGlyAspTyrVal                               657075                                                                         CTTAGCGTCTCCGAAAACTCGCGCGTCTCCCACTACATCATCAACAGC287                            LeuSerValSerGluAsnSerArgValSerHisTyrIleIleAsnSer                               80859095                                                                       AGCGGCCCGCGCCCTCCAGTGCCTCCGTCGCCCGCTCAGCCTCCGCCG335                            SerGlyProArgProProValProProSerProAlaGlnProProPro                               100105110                                                                      GGAGTGAGTCCCTCCAGGCTCCGAATAGGAGATCAAGAATTTGATTCA383                            GlyValSerProSerArgLeuArgIleGlyAspGlnGluPheAspSer                               115120125                                                                      TTGCCTGCTTTACTGGAATTCTACAAAATACACTATTTGGACACTACA431                            LeuProAlaLeuLeuGluPheTyrLysIleHisTyrLeuAspThrThr                               130135140                                                                      ACATTGATAGAACCAGTGGCCAGATCAAGGCAGGGTAGTGGAGTGATT479                            ThrLeuIleGluProValAlaArgSerArgGlnGlySerGlyValIle                               145150155                                                                      CTCAGGCAGGAGGAGGCAGAGTATGTGCGGGCCCTCTTTGACTTTAAT527                            LeuArgGlnGluGluAlaGluTyrValArgAlaLeuPheAspPheAsn                               160165170175                                                                   GGGAATGATGAAGAAGATCTTCCCTTTAAGAAAGGAGACATCCTGAGA575                            GlyAsnAspGluGluAspLeuProPheLysLysGlyAspIleLeuArg                               180185190                                                                      ATCCGGGATAAGCCTGAAGAGCAGTGGTGGAATGCAGAGGACAGCGAA623                            IleArgAspLysProGluGluGlnTrpTrpAsnAlaGluAspSerGlu                               195200205                                                                      GGAAAGAGGGGGATGATTCCTGTCCCTTACGTGGAGAAGTATAGACCT671                            GlyLysArgGlyMetIleProValProTyrValGluLysTyrArgPro                               210215220                                                                      GCCTCCGCCTCAGTATCGGCTCTGATTGGAGGTAACCAGGAGGGTTCC719                            AlaSerAlaSerValSerAlaLeuIleGlyGlyAsnGlnGluGlySer                               225230235                                                                      CACCCACAGCCACTGGGTGGCCGGAGCCTGGGCCCTATGCCAACCCAG767                            HisProGlnProLeuGlyGlyArgSerLeuGlyProMetProThrGln                               240245250255                                                                   CGT770                                                                         Arg                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 256 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        ProAspThrGlyAlaGlyProLeuGlyAlaGlyAlaArgAlaGlyGly                               151015                                                                         AlaArgValProAlaAlaAlaGlnArgGluSerAlaGluAlaAlaMet                               202530                                                                         AlaGlyAsnPheAspSerGluGluArgSerSerTrpTyrTrpGlyArg                               354045                                                                         LeuSerArgGlnGluAlaValAlaLeuLeuGlnGlyGlnArgAspGly                               505560                                                                         ValPheLeuValArgAspSerSerThrSerProGlyAspTyrValLeu                               65707580                                                                       SerValSerGluAsnSerArgValSerHisTyrIleIleAsnSerSer                               859095                                                                         GlyProArgProProValProProSerProAlaGlnProProProGly                               100105110                                                                      ValSerProSerArgLeuArgIleGlyAspGlnGluPheAspSerLeu                               115120125                                                                      ProAlaLeuLeuGluPheTyrLysIleHisTyrLeuAspThrThrThr                               130135140                                                                      LeuIleGluProValAlaArgSerArgGlnGlySerGlyValIleLeu                               145150155160                                                                   ArgGlnGluGluAlaGluTyrValArgAlaLeuPheAspPheAsnGly                               165170175                                                                      AsnAspGluGluAspLeuProPheLysLysGlyAspIleLeuArgIle                               180185190                                                                      ArgAspLysProGluGluGlnTrpTrpAsnAlaGluAspSerGluGly                               195200205                                                                      LysArgGlyMetIleProValProTyrValGluLysTyrArgProAla                               210215220                                                                      SerAlaSerValSerAlaLeuIleGlyGlyAsnGlnGluGlySerHis                               225230235240                                                                   ProGlnProLeuGlyGlyArgSerLeuGlyProMetProThrGlnArg                               245250255                                                                      (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1072 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 79..729                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        GCCAGTGAATTCGGGGGCTCAGCCCTCCTCCCTCCCTTCCCCCTGCTTCAGGCTGCTGAG60                 CACTGAGCAGCGCTCAGAATGGAAGCCATCGCCAAATATGACTTCAAAGCT111                         MetGluAlaIleAlaLysTyrAspPheLysAla                                              1510                                                                           ACTGCAGACGACGAGCTGAGCTTCAAAAGGGGGGACATCCTCAAGGTT159                            ThrAlaAspAspGluLeuSerPheLysArgGlyAspIleLeuLysVal                               152025                                                                         TTGAACGAAGAATGTGATCAGAACTGGTACAAGGCAGAGCTTAATGGA207                            LeuAsnGluGluCysAspGlnAsnTrpTyrLysAlaGluLeuAsnGly                               303540                                                                         AAAGACGGCTTCATTCCCAAGAACTACATAGAAATGAAACCACATCCG255                            LysAspGlyPheIleProLysAsnTyrIleGluMetLysProHisPro                               455055                                                                         TGGTTTTTTGGCAAAATCCCCAGAGCCAAGGCAGAAGAAATGCTTAGC303                            TrpPhePheGlyLysIleProArgAlaLysAlaGluGluMetLeuSer                               60657075                                                                       AAACAGCGGCACGATGGGGCCTTTCTTATCCGAGAGAGTGAGAGCGCT351                            LysGlnArgHisAspGlyAlaPheLeuIleArgGluSerGluSerAla                               808590                                                                         CCTGGGGACTTCTCCCTCTCTGTCAAGTTTGGAAACGATGTGCAGCAC399                            ProGlyAspPheSerLeuSerValLysPheGlyAsnAspValGlnHis                               95100105                                                                       TTCAAGGTGCTCCGAGATGGAGCCGGGAAGTACTTCCTCTGGGTGGTG447                            PheLysValLeuArgAspGlyAlaGlyLysTyrPheLeuTrpValVal                               110115120                                                                      AAGTTCAATTCTTTGAATGAGCTGGTGGATTATCACAGATCTACATCT495                            LysPheAsnSerLeuAsnGluLeuValAspTyrHisArgSerThrSer                               125130135                                                                      GTCTCCAGAAACCAGCAGATATTCCTGCGGGACATAGAACAGGTGCCA543                            ValSerArgAsnGlnGlnIlePheLeuArgAspIleGluGlnValPro                               140145150155                                                                   CAGCAGCCGACATACGTCCAGGCCCTCTTTGACTTTGATCCCCAGGAG591                            GlnGlnProThrTyrValGlnAlaLeuPheAspPheAspProGlnGlu                               160165170                                                                      GATGGAGAGCTGGGCTTCCGCCGGGGAGATTTTATCCATGTCATGGAT639                            AspGlyGluLeuGlyPheArgArgGlyAspPheIleHisValMetAsp                               175180185                                                                      AACTCAGACCCCAACTGGTGGAAAGGAGCTTGCCACGGGCAGACCGGC687                            AsnSerAspProAsnTrpTrpLysGlyAlaCysHisGlyGlnThrGly                               190195200                                                                      ATGTTTCCCCGCAATTATGTCACCCCCGTGAACCGGAACGTC729                                  MetPheProArgAsnTyrValThrProValAsnArgAsnVal                                     205210215                                                                      TAAGAGTCAAGAAGCAATTATTTAAAGAAAGTGAAAAATGTAAAACACATACAAAAGAAT789                TAAACCCACAAGCTGCCTCTGACAGCAGCCTGTGAGGGAGTGCAGAACACCTGGCCGGGT849                CACCCTGTGACCCTCTCACTTTGGTTGGAACTTTAGGGGGTGGGAGGGGGCGTTGGATTT909                AAAAATGCCAAAACTTACCTATAAATTAAGAAGAGTTTTTATTACAAATTTTCACTGCTG969                CTCCTCTTTCCCCTCCTTTGTCTTTTTTTTCATCCTTTTTTCTCTTCTGTCCATCAGTGC1029               ATGACGTTTAAGGCCACGTATAGTCCTAGCTGACGCCAATAAT1072                                (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 217 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetGluAlaIleAlaLysTyrAspPheLysAlaThrAlaAspAspGlu                               151015                                                                         LeuSerPheLysArgGlyAspIleLeuLysValLeuAsnGluGluCys                               202530                                                                         AspGlnAsnTrpTyrLysAlaGluLeuAsnGlyLysAspGlyPheIle                               354045                                                                         ProLysAsnTyrIleGluMetLysProHisProTrpPhePheGlyLys                               505560                                                                         IleProArgAlaLysAlaGluGluMetLeuSerLysGlnArgHisAsp                               65707580                                                                       GlyAlaPheLeuIleArgGluSerGluSerAlaProGlyAspPheSer                               859095                                                                         LeuSerValLysPheGlyAsnAspValGlnHisPheLysValLeuArg                               100105110                                                                      AspGlyAlaGlyLysTyrPheLeuTrpValValLysPheAsnSerLeu                               115120125                                                                      AsnGluLeuValAspTyrHisArgSerThrSerValSerArgAsnGln                               130135140                                                                      GlnIlePheLeuArgAspIleGluGlnValProGlnGlnProThrTyr                               145150155160                                                                   ValGlnAlaLeuPheAspPheAspProGlnGluAspGlyGluLeuGly                               165170175                                                                      PheArgArgGlyAspPheIleHisValMetAspAsnSerAspProAsn                               180185190                                                                      TrpTrpLysGlyAlaCysHisGlyGlnThrGlyMetPheProArgAsn                               195200205                                                                      TyrValThrProValAsnArgAsnVal                                                    210215                                                                         (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 642 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..471                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        GTGATTGAGAAGCCGGAGAATGACCCTGAATGGTGGAAATGCAAAAAT48                             ValIleGluLysProGluAsnAspProGluTrpTrpLysCysLysAsn                               151015                                                                         GCCCGAGGCCAAGTGGGCCTGGTCCCCAAAAACTACGTGGTTGTTCTC96                             AlaArgGlyGlnValGlyLeuValProLysAsnTyrValValValLeu                               202530                                                                         AGTGATGGGCCTGCTCTGCACCCCGCTCACACCCCCCAGATCAGCTAC144                            SerAspGlyProAlaLeuHisProAlaHisThrProGlnIleSerTyr                               354045                                                                         ACCGGGCCTTCAGCCAGCGGGCGCTTTGCTGGTCGGGAGTGGTACTAT192                            ThrGlyProSerAlaSerGlyArgPheAlaGlyArgGluTrpTyrTyr                               505560                                                                         GGCAACGTGACACGGCACCAGGCCGAGTGTGCGCTCAATGAGCGGGGC240                            GlyAsnValThrArgHisGlnAlaGluCysAlaLeuAsnGluArgGly                               65707580                                                                       GTCGAGGGCGACTTCCTCATTAGGGACAGCGAGTCCTCGCCCAGTGAC288                            ValGluGlyAspPheLeuIleArgAspSerGluSerSerProSerAsp                               859095                                                                         TTCTCCGTGTCTCTCAAAGCGTCAGGGAGAAACAAGCACTTCAAGGTG336                            PheSerValSerLeuLysAlaSerGlyArgAsnLysHisPheLysVal                               100105110                                                                      CAGCTGGTGGACAGCGTCTACTGCATTGGGCAGCGGCGGTTCCACAGC384                            GlnLeuValAspSerValTyrCysIleGlyGlnArgArgPheHisSer                               115120125                                                                      ATGGACGAGCTTGTGGAGCACTACAAGAAGGCCCCCATCTTCACCAGC432                            MetAspGluLeuValGluHisTyrLysLysAlaProIlePheThrSer                               130135140                                                                      GAGCACGGGGAGAAGCTCTACCTTGTCCGAGCCCTACAGTGAAAGCAGC481                           GluHisGlyGluLysLeuTyrLeuValArgAlaLeuGln                                        145150155                                                                      CATTGGCCCCCTCATGCCCTGCCCACTGTGGGCCTCGCTGCCACCTCTGCCTCCCAGAGC541                CCAGCACTTCTGGCCACCTCCACCCATGTGGCTTGGATCACCTCTGTGGCCCAGTCTGTC601                CTTTCTTTTTCAGCCCTGTTGGTCAACCACGGCTACCTAGG642                                   (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 157 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        ValIleGluLysProGluAsnAspProGluTrpTrpLysCysLysAsn                               151015                                                                         AlaArgGlyGlnValGlyLeuValProLysAsnTyrValValValLeu                               202530                                                                         SerAspGlyProAlaLeuHisProAlaHisThrProGlnIleSerTyr                               354045                                                                         ThrGlyProSerAlaSerGlyArgPheAlaGlyArgGluTrpTyrTyr                               505560                                                                         GlyAsnValThrArgHisGlnAlaGluCysAlaLeuAsnGluArgGly                               65707580                                                                       ValGluGlyAspPheLeuIleArgAspSerGluSerSerProSerAsp                               859095                                                                         PheSerValSerLeuLysAlaSerGlyArgAsnLysHisPheLysVal                               100105110                                                                      GlnLeuValAspSerValTyrCysIleGlyGlnArgArgPheHisSer                               115120125                                                                      MetAspGluLeuValGluHisTyrLysLysAlaProIlePheThrSer                               130135140                                                                      GluHisGlyGluLysLeuTyrLeuValArgAlaLeuGln                                        145150155                                                                      (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2345 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 369..1973                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        CTCTCTCTCTCTCTCTCTCTCCCTCTCTCCTAGCACCTGCTGCTCAGTAGGAAGGGCAAG60                 AGCAATTCGAGGCCGGTGCATTGTGAGGAGTCTCCACCCCTCCTCCTGCGCTTCCTTCTC120                CAGGGAGCCTCTCAGGCCGCCCTCACCTGCCCGAGATAATTTTAGTTTCCCTGGGCCTGG180                AATCTGGATACGCAGGGCCTCGCTCTATATTCTCCCGCCTCAACATTCCAAAGGCGGGAT240                AGCCTTTCTACCATCTGTAGAGAAGAGAGAAAGGATTCGAAATCAAATCCAAGTGTCTGG300                GATCTCTAGACAGAGCCAGACTTTGGGCCGGGTGTCCGGCTCCTTCTGTTGGAGGTGCTC360                CAGGTGCCATGGAACTGGATCTGAGCCCGACTCATCTCAGCAGCTCCCCA410                          MetGluLeuAspLeuSerProThrHisLeuSerSerSerPro                                     1510                                                                           GAAGATGTGTGCCCAACTCCTGCTACCCCTCCTGAGACTCCTCCGCCC458                            GluAspValCysProThrProAlaThrProProGluThrProProPro                               15202530                                                                       CCTGATAACCCTCCGCCAGGGGATGTGAAGCGGTCGCAGCCTTTGCCC506                            ProAspAsnProProProGlyAspValLysArgSerGlnProLeuPro                               354045                                                                         ATCCCCAGCAGCAGGAAACTTCGAGAAGAGGAGTTTCAGGCAACCTCT554                            IleProSerSerArgLysLeuArgGluGluGluPheGlnAlaThrSer                               505560                                                                         CTGCCCTCCATCCCCAACCCCTTCCCTGAGCTCTGCAGCCCACCTTCA602                            LeuProSerIleProAsnProPheProGluLeuCysSerProProSer                               657075                                                                         CAGAAACCCATTCTTGGTGGTTCCTCCGGTGCAAGGGGGTTGCTTCCT650                            GlnLysProIleLeuGlyGlySerSerGlyAlaArgGlyLeuLeuPro                               808590                                                                         CGAGACTCCAGCCGCCTCTGTGTGGTGAAGGTGTACAGTGAGGATGGG698                            ArgAspSerSerArgLeuCysValValLysValTyrSerGluAspGly                               95100105110                                                                    GCCTGCCGGTCTGTGGAGGTGGCAGCGGGCGCCACAGCTCGTCACGTG746                            AlaCysArgSerValGluValAlaAlaGlyAlaThrAlaArgHisVal                               115120125                                                                      TGTGAGATGCTGGTACAACGAGCTCACGCCCTGAGCGACGAGAGCTGG794                            CysGluMetLeuValGlnArgAlaHisAlaLeuSerAspGluSerTrp                               130135140                                                                      GGACTAGTGGAATCCCACCCCTACCTGGCACTGGAGCGGGGTCTGGAG842                            GlyLeuValGluSerHisProTyrLeuAlaLeuGluArgGlyLeuGlu                               145150155                                                                      GACCATGAATTTGTGGTGGAAGTGCAGGAGGCCTGGCCTGTGGGTGGA890                            AspHisGluPheValValGluValGlnGluAlaTrpProValGlyGly                               160165170                                                                      GATAGCCGCTTCATCTTCCGTAAAAACTTCGCCAAGTATGAACTATTC938                            AspSerArgPheIlePheArgLysAsnPheAlaLysTyrGluLeuPhe                               175180185190                                                                   AAGAGCCCCCCACACACCCTGTTTCCAGAAAAGATGGTCTCGAGCTGT986                            LysSerProProHisThrLeuPheProGluLysMetValSerSerCys                               195200205                                                                      CTGGATGCACAAACAGGCATATCCCATGAAGACCTCATCCAGAACTTC1034                           LeuAspAlaGlnThrGlyIleSerHisGluAspLeuIleGlnAsnPhe                               210215220                                                                      CTGAACGCTGGCAGCTTCCCTGAGATCCAGGGCTTCCTGCAGCTGCGG1082                           LeuAsnAlaGlySerPheProGluIleGlnGlyPheLeuGlnLeuArg                               225230235                                                                      GGATCAGGCCGGGGGTCAGGTCGAAAGCTTTGGAAACGTTTCTTCTGC1130                           GlySerGlyArgGlySerGlyArgLysLeuTrpLysArgPhePheCys                               240245250                                                                      TTTCTGCGTCGATCTGGCCTCTACTACTCTACCAAGGGTACCTCCAAG1178                           PheLeuArgArgSerGlyLeuTyrTyrSerThrLysGlyThrSerLys                               255260265270                                                                   GACCCCAGACACCTACAGTATGTGGCAGATGTGAATGAGTCCAATGTC1226                           AspProArgHisLeuGlnTyrValAlaAspValAsnGluSerAsnVal                               275280285                                                                      TATGTGGTGACCCAGGGCCGCAAGCTGTATGGGATGCCCACTGACTTC1274                           TyrValValThrGlnGlyArgLysLeuTyrGlyMetProThrAspPhe                               290295300                                                                      GGCTTCTGTGTCAAGCCCAACAAGCTTCGAAACGGCCACAAGGGGCTC1322                           GlyPheCysValLysProAsnLysLeuArgAsnGlyHisLysGlyLeu                               305310315                                                                      CACATCTTCTGCAGTGAGGATGAGCAGACTCGGACCTGCTGGCTGGCT1370                           HisIlePheCysSerGluAspGluGlnThrArgThrCysTrpLeuAla                               320325330                                                                      GCCTTCCGGCTCTTCAAGTACGGGGTACAGCTATATAAGAATTATCAG1418                           AlaPheArgLeuPheLysTyrGlyValGlnLeuTyrLysAsnTyrGln                               335340345350                                                                   CAGGCCCAGTCTCGTCACCTGCGCCTATCCTATTTGGGGTCTCCACCC1466                           GlnAlaGlnSerArgHisLeuArgLeuSerTyrLeuGlySerProPro                               355360365                                                                      TTGAGGAGCGTCTCAGACAATACCCTAGTGGCTATGGACTTCTCTGGC1514                           LeuArgSerValSerAspAsnThrLeuValAlaMetAspPheSerGly                               370375380                                                                      CATGCGGGGCGTGTCATTGATAACCCCCGGGAAGCTCTGAGTGCCGCC1562                           HisAlaGlyArgValIleAspAsnProArgGluAlaLeuSerAlaAla                               385390395                                                                      ATGGAGGAGGCCCAGGCCTGGAGGAAGAAGACAAACCACCGTCTGAGC1610                           MetGluGluAlaGlnAlaTrpArgLysLysThrAsnHisArgLeuSer                               400405410                                                                      CTGCCCACCACATGCTCTGGCTCGAGCCTCAGCGCAGCCATTCATCGC1658                           LeuProThrThrCysSerGlySerSerLeuSerAlaAlaIleHisArg                               415420425430                                                                   ACCCAGCCCTGGTTTCATGGACGCATCTCTCGGGAGGAGAGCCAGCGG1706                           ThrGlnProTrpPheHisGlyArgIleSerArgGluGluSerGlnArg                               435440445                                                                      CTAATTGGACAGCAGGGCCTGGTGGATGGTGTGTTCCTGGTCCGGGAG1754                           LeuIleGlyGlnGlnGlyLeuValAspGlyValPheLeuValArgGlu                               450455460                                                                      AGCCAGAGGAACCCACAGGGCTTTGTCCTGTCCTTGTGCCATCTGCAG1802                           SerGlnArgAsnProGlnGlyPheValLeuSerLeuCysHisLeuGln                               465470475                                                                      AAAGTCAAGCATTATCTCATTTTGCCAAGTGAAGATGAAGGTTGCCTT1850                           LysValLysHisTyrLeuIleLeuProSerGluAspGluGlyCysLeu                               480485490                                                                      TACTTCAGCATGGATGAGGGCCAGACCCGTTTCACAGACCTGCTGCAG1898                           TyrPheSerMetAspGluGlyGlnThrArgPheThrAspLeuLeuGln                               495500505510                                                                   CTGGTAGAATTCCACCAGCTGAACCGAGGCATCCTGCCCTGCCTGCTG1946                           LeuValGluPheHisGlnLeuAsnArgGlyIleLeuProCysLeuLeu                               515520525                                                                      CGCCACTGCTGTGCCCGTGTGGCCCTCTGAGGCCGCACAAGCTACTG1993                            ArgHisCysCysAlaArgValAlaLeu                                                    530535                                                                         CAGCCATGGGTTTGCCTACCACCCTTCTGTCCTGTGGACTCGGTGCAGGTGGGTGGGGTG2053               GTAAACAGTGGAAGAGCTCCCCCCCCCAATTTTATCCCATTTTTTTTAACCTCTCTCAAC2113               CAGTGAAACATCCCCTAACCCTGTCCATCCCTGACTCCTGTCCCCAAGGGAGGCATTGTG2173               GTCCTGTCCCCTTGGTAGAGCTCCTGAGGTACTGTTCCAGTGAGGGGCATTATGAGAGGA2233               GCGGGGCAGCCCAGGAGGTCTCATACCCCACCCATAATCTGTACAGACTGAGAGGCCAGT2293               TGATCTGCTCTGTTTTATACCAGTAACAATAAAGATTATTTTTTGATACAAA2345                       (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 535 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       MetGluLeuAspLeuSerProThrHisLeuSerSerSerProGluAsp                               151015                                                                         ValCysProThrProAlaThrProProGluThrProProProProAsp                               202530                                                                         AsnProProProGlyAspValLysArgSerGlnProLeuProIlePro                               354045                                                                         SerSerArgLysLeuArgGluGluGluPheGlnAlaThrSerLeuPro                               505560                                                                         SerIleProAsnProPheProGluLeuCysSerProProSerGlnLys                               65707580                                                                       ProIleLeuGlyGlySerSerGlyAlaArgGlyLeuLeuProArgAsp                               859095                                                                         SerSerArgLeuCysValValLysValTyrSerGluAspGlyAlaCys                               100105110                                                                      ArgSerValGluValAlaAlaGlyAlaThrAlaArgHisValCysGlu                               115120125                                                                      MetLeuValGlnArgAlaHisAlaLeuSerAspGluSerTrpGlyLeu                               130135140                                                                      ValGluSerHisProTyrLeuAlaLeuGluArgGlyLeuGluAspHis                               145150155160                                                                   GluPheValValGluValGlnGluAlaTrpProValGlyGlyAspSer                               165170175                                                                      ArgPheIlePheArgLysAsnPheAlaLysTyrGluLeuPheLysSer                               180185190                                                                      ProProHisThrLeuPheProGluLysMetValSerSerCysLeuAsp                               195200205                                                                      AlaGlnThrGlyIleSerHisGluAspLeuIleGlnAsnPheLeuAsn                               210215220                                                                      AlaGlySerPheProGluIleGlnGlyPheLeuGlnLeuArgGlySer                               225230235240                                                                   GlyArgGlySerGlyArgLysLeuTrpLysArgPhePheCysPheLeu                               245250255                                                                      ArgArgSerGlyLeuTyrTyrSerThrLysGlyThrSerLysAspPro                               260265270                                                                      ArgHisLeuGlnTyrValAlaAspValAsnGluSerAsnValTyrVal                               275280285                                                                      ValThrGlnGlyArgLysLeuTyrGlyMetProThrAspPheGlyPhe                               290295300                                                                      CysValLysProAsnLysLeuArgAsnGlyHisLysGlyLeuHisIle                               305310315320                                                                   PheCysSerGluAspGluGlnThrArgThrCysTrpLeuAlaAlaPhe                               325330335                                                                      ArgLeuPheLysTyrGlyValGlnLeuTyrLysAsnTyrGlnGlnAla                               340345350                                                                      GlnSerArgHisLeuArgLeuSerTyrLeuGlySerProProLeuArg                               355360365                                                                      SerValSerAspAsnThrLeuValAlaMetAspPheSerGlyHisAla                               370375380                                                                      GlyArgValIleAspAsnProArgGluAlaLeuSerAlaAlaMetGlu                               385390395400                                                                   GluAlaGlnAlaTrpArgLysLysThrAsnHisArgLeuSerLeuPro                               405410415                                                                      ThrThrCysSerGlySerSerLeuSerAlaAlaIleHisArgThrGln                               420425430                                                                      ProTrpPheHisGlyArgIleSerArgGluGluSerGlnArgLeuIle                               435440445                                                                      GlyGlnGlnGlyLeuValAspGlyValPheLeuValArgGluSerGln                               450455460                                                                      ArgAsnProGlnGlyPheValLeuSerLeuCysHisLeuGlnLysVal                               465470475480                                                                   LysHisTyrLeuIleLeuProSerGluAspGluGlyCysLeuTyrPhe                               485490495                                                                      SerMetAspGluGlyGlnThrArgPheThrAspLeuLeuGlnLeuVal                               500505510                                                                      GluPheHisGlnLeuAsnArgGlyIleLeuProCysLeuLeuArgHis                               515520525                                                                      CysCysAlaArgValAlaLeu                                                          530535                                                                         (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 6                                                                (D) OTHER INFORMATION: /label= A                                               /note= "The tyrosine is phosphorylated."                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       GluGluGluGluGluTyrMetProMetXaaXaa                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 6                                                                (D) OTHER INFORMATION: /label= A                                               /note= "The tyrosine is phosphorylated."                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       GluGluGluGluGluTyrValProMetXaaXaa                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 6                                                                (D) OTHER INFORMATION: /label= A                                               /note= "The tyrosine is phosphorylated."                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       AspAspAspAspAspTyrMetProMetXaaXaa                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 6                                                                (D) OTHER INFORMATION: /label= A                                               /note= "The tyrosine is phosphorylated."                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       AspAspAspAspAspTyrValProMetXaaXaa                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       IleGluGlyArg                                                                   1                                                                              (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       LeuValProArg                                                                   1                                                                              (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 96 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       TrpTyrTrpGlyAspIleSerArgGluGluValAsnGluLysLeuArg                               151015                                                                         AspThrAlaAspGlyThrPheLeuValArgAspSerThrLysMetHis                               202530                                                                         GlyAspTyrThrLeuThrLeuArgLysGlyGlyAsnAsnLeuIleLys                               354045                                                                         IlePheHisArgAspGlyLysTyrGlyPheSerAspProLeuThrPhe                               505560                                                                         SerSerValValGluLeuIleAsnHisTyrArgAsnGluSerLeuAla                               65707580                                                                       GlnTyrAsnProLysLeuAspValLysLeuLeuTyrProValSerLys                               859095                                                                         (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 97 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       TrpAsnValGlySerSerAsnArgAsnLysAlaGluAsnLeuLeuArg                               151015                                                                         GlyLysArgAspGlyThrPheLeuValArgGluSerSerLysGlnGly                               202530                                                                         CysTyrAlaCysSerValValValAspGlyGluValLysHisCysVal                               354045                                                                         IleAsnLysThrAlaThrGlyTyrGlyPheAlaGluProTyrAsnLeu                               505560                                                                         TyrSerSerLeuLysGluLeuValLeuHisTyrGlnHisThrSerLeu                               65707580                                                                       ValGlnHisAsnAspSerLeuAsnValThrLeuAlaTyrProValTyr                               859095                                                                         Ala                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 99 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       TrpTyrPheGlyLysIleThrArgArgGluSerGluArgLeuLeuLeu                               151015                                                                         AsnProGluAsnProArgGlyThrPheLeuValArgGluSerGluThr                               202530                                                                         ThrLysGlyAlaTyrCysLeuSerValSerAspPheAspAsnAlaLys                               354045                                                                         GlyLeuAsnValLysHisTyrLysIleArgLysLeuAspSerGlyGly                               505560                                                                         PheTyrIleThrSerArgThrGlnPheSerSerLeuGlnGlnLeuVal                               65707580                                                                       AlaTyrTyrSerLysHisAlaAspGlyLeuCysHisArgLeuThrAsn                               859095                                                                         ValCysPro                                                                      (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 92 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       TrpTyrHisGlyProValSerArgAsnAlaAlaGluTyrLysLysSer                               151015                                                                         SerGlyIleAsnGlySerPheLeuValArgGluSerGluSerSerPro                               202530                                                                         GlyGlnArgSerIleSerLeuArgTyrGluGlyArgValTyrHisTyr                               354045                                                                         ArgIleAsnThrAlaSerAspGlyLysLeuTyrValSerSerGluSer                               505560                                                                         ArgPheAsnThrLeuAlaGluLeuValHisHisHisSerThrValAla                               65707580                                                                       AspGlyLeuIleThrThrLeuHisTyrProAlaPro                                           8590                                                                           (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 109 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       TrpPheHisGlyLysLeuGlyAlaGlyArgAspGlyArgHisIleAla                               151015                                                                         GluArgLeuLeuThrGluTyrCysIleGluThrGlyAlaProAspGly                               202530                                                                         SerPheLeuValArgGluSerGluThrPheValGlyAspTyrThrLeu                               354045                                                                         SerPheTrpArgAsnGlyLysValGlnHisCysArgIleHisSerArg                               505560                                                                         GlnAspAlaGlyThrProLysPhePheLeuThrAspAsnLeuValPhe                               65707580                                                                       AspSerLeuTyrAspLeuIleThrHisTyrGlnGlnValProLeuArg                               859095                                                                         CysAlaGluPheGluMetArgLeuSerLeuProValPro                                        100105                                                                         (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 89 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       TrpTyrHisAlaSerLeuThrArgAlaGlnAlaGluHisMetLeuMet                               151015                                                                         ArgValProArgAspGlyAlaPheLeuValArgLysArgAsnGluPro                               202530                                                                         AsnSerTyrAlaIleSerPheArgAlaGluGlyLysIleLysHisCys                               354045                                                                         ArgValGlnGlnGluGlyGlnThrValMetLeuGlyAsnSerGluPhe                               505560                                                                         AspSerLeuValAspLeuIleSerTyrTyrGluLysHisProLeuTyr                               65707580                                                                       ArgLysMetLysLeuArgTyrProIle                                                    85                                                                             (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 94 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       TrpTyrHisGlyLysLeuAspArgThrIleAlaGluGluArgLeuArg                               151015                                                                         GlnAlaGlyLysSerGlySerTyrLeuIleArgGluSerAspArgArg                               202530                                                                         ProGlySerPheValLeuSerPheArgSerGlnMetAsnValValAsn                               354045                                                                         HisProArgIleIleAlaMetCysGlyAspTyrTyrIleGlyGlyArg                               505560                                                                         ArgPheSerSerLeuSerAspLeuIleGlyTyrTyrSerHisValSer                               65707580                                                                       CysLeuLeuLysGlyGluLysLeuLeuTyrProValAlaPro                                     8590                                                                           (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 92 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       TrpTyrHisGlyLysIleSerLysGlnGluAlaTyrAsnLeuLeuMet                               151015                                                                         ThrValGlyGlnValCysSerPheLeuValArgProSerAspAsnThr                               202530                                                                         ProGlyAspTyrSerLeuTyrPheArgThrAsnGluAsnIleGlnArg                               354045                                                                         PheLysIleCysProThrProAsnAsnGlnPheMetMetGlyGlyArg                               505560                                                                         TyrTyrAsnSerIleGlyAspIleIleAspHisTyrArgLysGluGln                               65707580                                                                       IleValGluGlyTyrTyrLeuLysGluProValPro                                           8590                                                                           (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 107 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       TrpTyrTrpGlyArgLeuSerArgGlyAspAlaValSerLeuLeuGln                               151015                                                                         GlyGlnArgHisGlyThrPheLeuValArgAspSerGlySerIlePro                               202530                                                                         GlyAspPheValLeuSerValSerGluSerSerArgValSerHisTyr                               354045                                                                         IleValAsnSerLeuGlyProAlaGlyGlyArgArgAlaGlyGlyGlu                               505560                                                                         GlyProPheAlaProGlyLeuAsnProThrArgPheLeuIleGlyAsp                               65707580                                                                       AsnValPheAspSerLeuProSerLeuLeuGluPheTyrLysIleHis                               859095                                                                         TyrLeuAspThrThrThrLeuIleGluProVal                                              100105                                                                         (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 70 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       AlaLeuTyrAspTyrLysLysGluArgGluGluAspIleAspLeuHis                               151015                                                                         LeuGlyAspIleLeuThrValAsnLysGlySerLeuValAlaLeuGly                               202530                                                                         PheSerAspProGluAlaArgProGluAspIleGlyTrpLeuAsnGly                               354045                                                                         TyrAsnGluThrThrGlyGluArgGlyAspPheProGlyThrTyrVal                               505560                                                                         GluTyrIleGlyArgLys                                                             6570                                                                           (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 55 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       AlaLeuTyrAspTyrGluSerArgThrGluThrAspLeuAlaPheLys                               151015                                                                         LysGlyGluArgLeuGlnIleValMetAsnThrGluGlyAspTrpTrp                               202530                                                                         LeuAlaHisSerLeuThrThrGlyGlnThrGlyTyrIleProSerAsn                               354045                                                                         TyrValAlaProSerAspSer                                                          5055                                                                           (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 54 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       AlaLeuTyrAspPheValAlaSerGlyAspAsnThrLeuSerIleThr                               151015                                                                         LysGlyGluLysLeuArgValLeuGlyTyrAsnHisAsnGlyGluTrp                               202530                                                                         CysGluAlaGlnThrLysAsnGlyGlnGlyTrpValProSerAsnTyr                               354045                                                                         IleThrProValAsnSer                                                             50                                                                             (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 54 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       AlaLeuPheAspTyrLysAlaGlyArgGluAspGluLeuThrPheThr                               151015                                                                         LysSerAlaIleIleGlnAsnValGluLysGlnGluGlyGlyTrpTrp                               202530                                                                         ArgGlyAspTyrHisHisLysLysGlnLeuTrpPheProSerAsnTyr                               354045                                                                         ValGluGluMetValSer                                                             50                                                                             (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       AlaIleLeuAspTyrThrLysValProAspThrAspGluIleSerPhe                               151015                                                                         LeuLysGlyAspMetPheIleValAsnAsnGluLeuGluAspGlyTrp                               202530                                                                         MetTrpValThrAsnLeuArgThrAspGluGlnGlyLeuIleValGlu                               354045                                                                         AspLeuValGluGluValGlyArg                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 55 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       AlaLeuPheAspPheLysGlyAsnAspAspGlyAspLeuProPheLys                               151015                                                                         LysGlyAspIleLeuLysIleArgAspLysProGluGluGlnTrpTrp                               202530                                                                         AsnAlaGluAspMetAspGlyLysArgGlyMetIleProValProTyr                               354045                                                                         ValGluLysCysArgProSer                                                          5055                                                                           (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 949 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..549                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 553..585                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 607..642                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 646..690                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 694..894                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 910..942                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       GCCAGTGAATTCGGGCCCGAATTGGCAGAGCTTAATGGAAAAGACGGCTTCATTCCCAAG60                 AACTACATAGAAATGAAACCACATCCGTGGTTTTTTGGCAAAATCCCCAGAGCCAAGGCA120                GAAGAAATGCTTAGCAAACAGCGGCACGATGGGGCCTTTCTTATCCGAGAGAGTGAGAGC180                GCTCCTGGGGACTTCTCCCTCTCTGTCAAGTTTGGAACGATGTGCAGCACTTTCAAGGTG240                CTCCCGAGATGGAGCCGGGAAGTACTTCCTCTGGTGGTGAAGTTCAATTCTTTGAATGAG300                CTGGTGGATTATCACAGATCTACATCTGTCTCCAGAAACCAGCAGATATTCCTGCGGGAC360                ATAGAACAGGTGCCACAGCAGCCGACATACGTCCAGGCCCTCTTTGACTTTGATCCCCAG420                GAGGATGGAGAGCTGGGCTTCCGCCGGGGAGATTTTATCCATGTCATGGATAACTCAGAC480                CCCAACTGGTGGAAAGGAGCTTGCCACGGGCAGACCGGCATGTTTCCCCGCGAATTATGT540                CTCCCCCNGTGAACCGGAACGTCTAAGAGTCAAGAAGCAATTATTTAAAGAAAGTGAAAA600                ATGTAAAACACATACAAAAGAATTAAACCCACAAGCTGCCTCTGACAGCAGCCTGTGAGG660                GAGTGCAGAACACCTGGCCGGGTCACCCTGTGACCCTCTCACTTTGGTTGGAACTTTAGG720                GGGTGGGAGGGGGCGTTGGATTTAAAAATGCCAAAACTTACCTATAAATTAAGAAGAGTT780                TTTATTACAAATTTTCACTGCTGCTCCTCTTTCCCCTCCTTTGTCTTTTTTTTTCATCCT840                TTTTTCTCTTCTGTCCATCAGTGCATGACGTTTAAGGCCACGTATAGTCCTAGCTGACGC900                CAATAATAAAAACCGAATTCGAGCTCGGATCCGGGGATCCTCTAGAGTC949                           (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 183 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       AlaSerGluPheGlyProGluLeuAlaGluLeuAsnGlyLysAspGly                               151015                                                                         PheIleProLysAsnTyrIleGluMetLysProHisProTrpPhePhe                               202530                                                                         GlyLysIleProArgAlaLysAlaGluGluMetLeuSerLysGlnArg                               354045                                                                         HisAspGlyAlaPheLeuIleArgGluSerGluSerAlaProGlyAsp                               505560                                                                         PheSerLeuSerValLysPheGlyThrMetCysSerThrPheLysVal                               65707580                                                                       LeuProArgTrpSerArgGluValLeuProLeuValValLysPheAsn                               859095                                                                         SerLeuAsnGluLeuValAspTyrHisArgSerThrSerValSerArg                               100105110                                                                      AsnGlnGlnIlePheLeuArgAspIleGluGlnValProGlnGlnPro                               115120125                                                                      ThrTyrValGlnAlaLeuPheAspPheAspProGlnGluAspGlyGlu                               130135140                                                                      LeuGlyPheArgArgGlyAspPheIleHisValMetAspAsnSerAsp                               145150155160                                                                   ProAsnTrpTrpLysGlyAlaCysHisGlyGlnThrGlyMetPhePro                               165170175                                                                      ArgGluLeuCysLeuProXaa                                                          180                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       ThrGlyThrSerLysSerGlnGluAlaIleIle                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:35:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                       AsnThrTyrLysArgIleLysProThrSerCysLeu                                           1510                                                                           (2) INFORMATION FOR SEQ ID NO:36:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                       GlnGlnProValArgGluCysArgThrProGlyArgValThrLeu                                  151015                                                                         (2) INFORMATION FOR SEQ ID NO:37:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 67 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                       ProSerHisPheGlyTrpAsnPheArgGlyTrpGluGlyAlaLeuAsp                               151015                                                                         LeuLysMetProLysLeuThrTyrLysLeuArgArgValPheIleThr                               202530                                                                         AsnPheHisCysCysSerSerPheProSerPheValPhePhePheHis                               354045                                                                         ProPhePheSerSerValHisGlnCysMetThrPheLysAlaThrTyr                               505560                                                                         SerProSer                                                                      65                                                                             (2) INFORMATION FOR SEQ ID NO:38:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                       LysProAsnSerSerSerAspProGlyIleLeu                                              1510                                                                           (2) INFORMATION FOR SEQ ID NO:39:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 236 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                       GlnProArgAlaGlyArgGlyAlaGlyHisArgGlyLeuArgArgPro                               151015                                                                         AlaGlyArgGlyGlnArgValArgProAlaGlyGlyAlaAlaLeuMet                               202530                                                                         AlaGlyGlnGluAspSerGluAspArgGlySerTrpTyrTrpGlyArg                               354045                                                                         LeuSerArgGlyAspAlaValSerLeuIleGlnGlyGlnArgHisGly                               505560                                                                         ThrPheLeuValArgAspSerGlySerIleProGlyAspPheValLeu                               65707580                                                                       SerValSerGluSerSerArgValSerHisTyrIleValAsnSerLeu                               859095                                                                         GlyProAlaGlyGlyArgArgAlaGlyGlyGluGlyProGlyAlaPro                               100105110                                                                      GlyLeuAsnProThrArgPheLeuIleGlyAspGlnValPheAspSer                               115120125                                                                      LeuProSerLeuLeuGluPheTyrLysIleHisTyrLeuAspThrThr                               130135140                                                                      ThrLeuIleGluProValSerArgSerArgGlnAsnSerGlyValIle                               145150155160                                                                   LeuArgGlnGluGluValGluTyrValArgAlaLeuPheAspPheLys                               165170175                                                                      GlyAsnAspAspGlyAspLeuProPheLysLysGlyAspIleLeuLys                               180185190                                                                      IleArgAspLysProGluGluGlnTrpTrpAsnAlaGluAspMetAsp                               195200205                                                                      GlyLysArgGlyMetIleProValProTyrValGluLysCysArgPro                               210215220                                                                      SerSerAlaSerValSerThrLeuThrGlyGlyArg                                           225230235                                                                      (2) INFORMATION FOR SEQ ID NO:40:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 160 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                       ValIleGluLysProGluAsnAspProGluTrpTrpLysCysArgLys                               151015                                                                         IleAsnGlyMetValGlyLeuValProLysAsnTyrValThrValMet                               202530                                                                         GlnAsnAsnProLeuThrSerGlyLeuGluProSerHisProProGln                               354045                                                                         CysAspTyrIleArgProSerLeuThrGlyLysPheAlaGlyAsnPro                               505560                                                                         TrpTyrTyrGlyLysValThrArgHisGlnAlaGluMetAlaLeuAsn                               65707580                                                                       GluArgGlyHisGluGlyAspPheLeuIleArgAspSerGluSerSer                               859095                                                                         ProAsnAspPheSerValSerLeuLysAlaGlnGlyLysAsnLysHis                               100105110                                                                      PheLysValGlnLeuLysGluThrValTyrCysIleGlyGlnArgLys                               115120125                                                                      PheSerThrMetGluGluLeuValGluHisTyrLysLysAlaProIle                               130135140                                                                      PheThrSerGluGlnGlyGluLysLeuTyrLeuValLysHisLeuSer                               145150155160                                                                   (2) INFORMATION FOR SEQ ID NO:41:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 96 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                       TrpTyrPheGlyLysLeuGlyArgLysAspAlaGluArgGlnLeuLeu                               151015                                                                         SerPheGlyAsnProArgGlyThrPheLeuIleArgGluSerGluThr                               202530                                                                         ThrLysGlyAlaTyrSerLeuSerIleArgAspTrpAspAspMetLys                               354045                                                                         GlyAspHisValLysHisTyrLysIleArgLysLeuAspAsnGlyGly                               505560                                                                         TyrTyrIleThrThrArgAlaGlnPheGluThrLeuGlnGlnLeuVal                               65707580                                                                       GlnHisTyrSerGluArgAlaAlaGlyLeuCysCysArgLeuValVal                               859095                                                                         (2) INFORMATION FOR SEQ ID NO:42:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 92 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                       LysGlyLysGlyLysArgTrpLysAsnLeuTyrPheIleLeuGluGly                               151015                                                                         SerAspAlaGlnLeuIleTyrPheGluSerGluLysArgAlaThrLys                               202530                                                                         ProLysGlyLeuIleAspLeuSerValCysSerValTyrValValHis                               354045                                                                         AspSerLeuPheGlyArgProAsnCysPheGlnIleValValGlnHis                               505560                                                                         PheSerGluGluHisTyrIlePheTyrPheAlaGlyGluThrProGlu                               65707580                                                                       GlnAlaGluAspTrpMetLysGlyLeuGlnAlaPhe                                           8590                                                                           (2) INFORMATION FOR SEQ ID NO:43:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 223 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                       ProLysProAlaArgAlaAlaProProProProProProProProPro                               151015                                                                         ProProGlyAlaAspArgValValLysAlaValProPheProProThr                               202530                                                                         HisArgLeuThrSerGluGluValPheAspLeuAspGlyIleProArg                               354045                                                                         ValAspValLeuLysAsnHisLeuValLysGluGlyArgValAspGlu                               505560                                                                         GluIleAlaLeuArgIleIleAsnGluGlyAlaAlaIleLeuArgArg                               65707580                                                                       GluLysThrMetIleGluValGluAlaProIleThrValCysGlyAsp                               859095                                                                         IleHisGlyGlnPhePheAspLeuMetLysLeuPheLysValGlyGly                               100105110                                                                      SerProAlaAsnThrArgTyrLeuPheLeuGlyAspTyrValAspArg                               115120125                                                                      GlyTyrPheSerIleGluCysValLeuTyrLeuTrpValLeuLysIle                               130135140                                                                      LeuTyrProSerThrLeuPheLeuLeuArgGlyAsnHisGluCysArg                               145150155160                                                                   HisLeuThrGluTyrPheThrPheLysGlnGluCysLysIleLysTyr                               165170175                                                                      SerGluArgValTyrGluAlaCysMetGluAlaPheAspSerLeuPro                               180185190                                                                      LeuAlaAlaLeuLeuAsnGlnGlnPheLeuCysValHisGlyGlyLeu                               195200205                                                                      SerProGluIleHisThrLeuAspAspIleArgArgLeuAspArg                                  210215220                                                                      (2) INFORMATION FOR SEQ ID NO:44:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 99 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                       TrpPhePheGlyLysIleProArgAlaLysAlaGluGluMetLeuSer                               151015                                                                         LysGlnArgHisAspGlyAlaPheLeuIleArgGluSerGluSerAla                               202530                                                                         ProGlyAspPheSerLeuSerValLysPheGlyAsnAspValGlnHis                               354045                                                                         PheLysValLeuArgAspGlyAlaGlyLysTyrPheLeuTrpValVal                               505560                                                                         LysPheAsnSerLeuAsnGluLeuValAspTyrHisArgSerThrSer                               65707580                                                                       ValSerArgAsnGlnGlnIlePheLeuArgAspIleGluGlnValPro                               859095                                                                         GlnGlnPro                                                                      (2) INFORMATION FOR SEQ ID NO:45:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 50 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                       AlaLysTyrAspPheLysAlaThrAlaAspAspGluLeuSerPheLys                               151015                                                                         ArgGlyAspIleLeuLysValLeuAsnGluGluCysAspGlnAsnTrp                               202530                                                                         TyrLysAlaGluLeuAsnGlyLysAspGlyPheIleProLysAsnTyr                               354045                                                                         IleGlu                                                                         50                                                                             (2) INFORMATION FOR SEQ ID NO:46:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 46 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                       AlaLeuPheAspPheAspProGlnGluAspGlyGluLeuGlyPheArg                               151015                                                                         ArgGlyAspPheIleHisValMetAspAsnSerAspProAsnTrpTrp                               202530                                                                         LysGlyAlaCysHisGlyGlnThrGlyMetPheProArgAsn                                     354045                                                                         (2) INFORMATION FOR SEQ ID NO:47:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 228 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                       MetGluAlaValAlaGluHisAspPheGlnAlaGlySerProAspGlu                               151015                                                                         LeuSerPheLysArgGlyAsnThrLeuLysValLeuAsnLysAspGlu                               202530                                                                         AspProHisTrpTyrLysAlaGluLeuAspGlyAsnGluGlyPheIle                               354045                                                                         ProSerAsnTyrIleArgMetThrGluCysAsnTrpTyrLeuGlyLys                               505560                                                                         IleThrArgAsnAspAlaGluValLeuLeuLysLysProThrValArg                               65707580                                                                       AspGlyHisPheLeuValArgGlnCysGluSerSerProGlyGluPhe                               859095                                                                         SerIleSerValArgPheGlnAspSerValGlnHisPheLysValLeu                               100105110                                                                      ArgAspGlnAsnGlyLysTyrTyrLeuTrpAlaValLysPheAsnSer                               115120125                                                                      LeuAsnGluLeuValAlaTyrHisArgThrAlaSerValSerArgThr                               130135140                                                                      HisThrIleLeuLeuSerAspMetAsnValGluThrLysPheValGln                               145150155160                                                                   AlaLeuPheAspPheAsnProGlnGluSerGlyGluLeuAlaPheLys                               165170175                                                                      ArgGlyAspValIleThrLeuIleAsnLysAspAspProAsnTrpTrp                               180185190                                                                      GluGlyGlnLeuAsnAsnArgArgGlyIlePheProSerAsnTyrVal                               195200205                                                                      CysProTyrAsnSerAsnLysSerAsnSerAsnValAlaProGlyPhe                               210215220                                                                      AsnPheGlyAsn                                                                   225                                                                            (2) INFORMATION FOR SEQ ID NO:48:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3372 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                       CCAGTGAATTCGGGTTTTTTTTTTATTGAATAAAGCAGCTTTGAAGCTTATCAGGCACTG60                 CCCAACAAAACAGTCCAAAAAAAATCTGTTATGAACTTTTTAACCTTGGGCTAACAGCAA120                GTACTCTGTACAAGAAAAACTGTAAAATATTTCATAGTTTTAATTCTGAAGCCATTTTTT180                TTTTTACTGGCATCCTGTACATTTATTTTTAAAAAAGGATAACAAAAATGAATATTAACA240                AAAATCCGGGACAACAATATTTTCAAGCAACAAAAACTGGGGTGGGGAAGCTTATTCTGA300                AGGTACATTTAAAACTGAAATAACAACTTAATGAAAATTAAGAATTGCATAGCGCTGTGA360                ATTTAGCCTTCAGCAAAACAAAACAGAAGCTATTTGGTATTGATACAAATCCATCTATTT420                GATAGTTAGTCATCCAATATTATGTACATATTTTATATACTGAATGTCATTTTAAGTCCT480                GTTTTCCAAACTCCATTTTTCTGTTGCTGGGTTTTTGTTTTTTGACAAGTTAAACACTTT540                CTGGCACTTTCTATGACAGAATTTCTTCTGAACATACATGAACTGACATTCTCCCAAAGC600                GTCCCTTGTGAGTGGACGTGCCTTTCTGCTACATATCGTTCATTTGTTACAAAATGAAAT660                AATCCACAGTGCGATGTGTCTGGGTCCACCGTGCACAGCAACATCCAGGCTAAACCAGGC720                TGGACCAAACCTTCTCAGGCCAGGGTTCTTCTGCCTCTCTCTCCTCCGCTCTCCAGAGTC780                CCAACTTTCAGCTGGTAAAGCTTCAGAAAGCATTAACAAGCACCATGGTTAAAGGCCTCT840                TTGTCCCTGCACGCAGAGATTTTTGCATTTCTTTTTCTCTTTGTGTTGTATGTGGTTGTG900                GCTTTAAATTAAACCAAAGAAAAAAAGAAAAAGAAGGGATAAGCACTCCAGCTTAGGAAC960                GTGTGGTCATCATACAGCCTTAGGCTGCATGTCTTCCCCTACTTCTTTTTTGTCAAAGAA1020               AGCTCTAGTCCCATCCAAAGAAAGATGGCTTCGTTTCTGCAGCTCAATTCACAGATCAGA1080               CTGGAGAGGAGCCCTTTGCTTTCCAGAGGCCTCAGGGTGGCTGAACTTCAGGAGAAGGAT1140               CAAAGAGTAAGCGCTTCATCGCCTCTGCTGTGCATATACTGGGTAGGCTAGTGTGACATT1200               GAGGGAGTCGGTGTGCTGCACAAGGGAGGTGTGTTGGTAATGTAGCACCAGTTCTTTCAG1260               AGAGCTGTACAAGTTATAGGGCTCGGCAAAGCCATAGCCAGTTGCTGTTTTGTTTATGAC1320               ACAATGCTTTACTTCGCCGTCCACCACTACAGAGCAGGCATAGCAGCCCTGTTTACTGCT1380               CTCCCGGACAAGAAAAGTGCCATCTCGCTTCCCTCGCAACAGGTTTTCAGCTTTGTTTCG1440               GTTGCTGCTTCCAACATTCCATGTCTTCTCATCATGATGGGGCAAATCTTCATCATCTTC1500               CACCAGTGAATATTGGTCTTCAGTGTTTTCATTGCCCAACCACTCGTTCAACTTCTTTTG1560               CCGAACACCTTTTTGAGTCAACCACATCAAGTATTGGTCTCTCGTCTTTCTCAGCTGGAT1620               AAGGTCTGGTTTAATGCTGTTCATACGTTTGTCAATTTCTCGATACTCAGCTGCCTGCTT1680               CTTCAAGTCTTCTTCCAATCTTCTTCTACTGTCAATAATTTCACTGATTCGAGACTTCAA1740               CTTATCATAATTATGCATAATCCTTTGTATTTCTTTCTCATTGCCTTCACGTTTAAACTT1800               TTCTATGTATTCTTTGCTGTACCGCTCTTGGGTCTGGCACTGTTCTTCAAATATTTTTAT1860               GGTTTCATTAAATGCTTCAATAGCTGTCCTTTTCATTTGGATTTCCTGGGATGTGCGGGT1920               ATATTCTTCATATAATCTATCATATTCTCGACTTTTTTCTTGAAACTGAGTGTTATATTC1980               ATGTAATTTTTTCCCTACAGCTTCAATATTATCTTCTTTGACAACTTGATCCTGTTGGTA2040               TTTGGATACTGGATAAAGTAATTTCACATCCAATTTGGGATTATACTGAGCTAGAGATTC2100               ATTCCGGTAGTGGTTTATTAATTCAACCACAGAACTGAAGGTTAATGGGTCAGAGAAGCC2160               ATATTTCCCATCTCGATGAAATATTTTGATTAATTTGTTATTTCCCCCTTTCCTTAGTGT2220               AAGAGTATAATCACCATGCATTTTAGTAGACGCATCTCGTACCAAAAAGGTCCCGTCTGC2280               TGTATCTCGAAGTTTTTCATTCACTTCTTCCCTCGAGATATCTCCCCAGTACCATTCAGC2340               ATTTTGTAAGGACATATTGTTATTCATACCGTTGTTGGCTACAGTAGTAGGTTTTGGTGG2400               TTTAGGAGGCAGTGCTGGTGCAGGCTGTCGTTCATTCCATTCAGTTGAGATTAAAATTTC2460               TATAACTTTTATGAGGTTTTCAGTATTATCAGAGCTGGCTGCTGAGAATCTGAAAAGCAT2520               AGGGCTGAAAATTTCAGAGAGTACTCTTGCATTCAACAGATTTTTGCTGGACGTTTGAGA2580               GAGCTTGAAGAAATGTTTTAACAAATACTGAAGCGTAAGCCAATACTGATGAGGTATGCT2640               AGGCGACCTAATAAGCTTCTTCAATAGCTGAATATATTCTTCGGAGCTTTGTACTTCTGG2700               AGCTAAAGAAATCATTTCACTGTAAACGGCTGCTGGAATGACAGGATTTGGTAAGTCCAG2760               GAGATAGCGTTTGAAAGCGTCAGCCAAAACGTGCACATCGATCATTTCCAAGTCCACGGA2820               GGGTGTATCACAATCAAGAAGCTGTCGTAATTCTGCCAGGTTGCTGGAGCTCTGTGTTCT2880               GTATAGAGTTGAACATTCCAGACCTTTCTTTTCAATGGCTTCCACGAGCTTGATAAGAAG2940               AGGCGGGGCAATGTCAGGAGGGGCAAACTGCTCTGCAAGATCCGGGAGAGTCAAAGCTTG3000               TTGTTCAACATCTGCTTCAGTTTTCGAAGAACCTGGTGCAACAGGAAGAGGCCGAGGTGG3060               CCGGGGCTTTGGTGTGGGAGGCGAGATTTTTTTCCTTCCAATATATTCTACGTAAGTTCC3120               CGGAAAGTCCCCCTTTTCCCCTGTGGTTTCATTATAGCCATTTAACCAGCCATTTCTTCG3180               AGGCCTGGCTTCCTGTCCATCACTGAATCCAAGAGCTACTAAGGACCCTTTATTCACAGT3240               CAATATGTCACCCAAGTGCAAGTCAATATCTTCTTCTCTTTCCTTTTTATAATCATACAG3300               CGCTCTGTACTGGTACCCCTCAGCACTCATGTTTGCAAATCTGCTACCATGCAACAGTTG3360               AGCCTGGTTGTA3372                                                               (2) INFORMATION FOR SEQ ID NO:49:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1072 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                       ATTATTGGCGTCAGCTAGGACTATACGTGGCCTTAAACGTCATGCACTGATGGACAGAAG60                 AGAAAAAAGGATGAAAAAAAAGACAAAGGAGGGGAAAGAGGAGCAGCAGTGAAAATTTGT120                AATAAAAACTCTTCTTAATTTATAGGTAAGTTTTGGCATTTTTAAATCCAACGCCCCCTC180                CCACCCCCTAAAGTTCCAACCAAAGTGAGAGGGTCACAGGGTGACCCGGCCAGGTGTTCT240                GCACTCCCTCACAGGCTGCTGTCAGAGGCAGCTTGTGGGTTTAATTCTTTTGTATGTGTT300                TTACATTTTTCACTTTCTTTAAATAATTGCTTCTTGACTCTTAGACGTTCCGGTTCACGG360                GGGTGACATAATTGCGGGGAAACATGCCGGTCTGCCCGTGGCAAGCTCCTTTCCACCAGT420                TGGGGTCTGAGTTATCCATGACATGGATAAAATCTCCCCGGCGGAAGCCCAGCTCTCCAT480                CCTCCTGGGGATCAAAGTCAAAGAGGGCCTGGACGTATGTCGGCTGCTGTGGCACCTGTT540                CTATGTCCCGCAGGAATATCTGCTGGTTTCTGGAGACAGATGTAGATCTGTGATAATCCA600                CCAGCTCATTCAAAGAATTGAACTTCACCACCCAGAGGAAGTACTTCCCGGCTCCATCTC660                GGAGCACCTTGAAGTGCTGCACATCGTTTCCAAACTTGACAGAGAGGGAGAAGTCCCCAG720                GAGCGCTCTCACTCTCTCGGATAAGAAAGGCCCCATCGTGCCGCTGTTTGCTAAGCATTT780                CTTCTGCCTTGGCTCTGGGGATTTTGCCAAAAAACCACGGATGTGGTTTCATTTCTATGT840                AGTTCTTGGGAATGAAGCCGTCTTTTCCATTAAGCTCTGCCTTGTACCAGTTCTGATCAC900                ATTCTTCGTTCAAAACCTTGAGGATGTCCCCCCTTTTGAAGCTCAGCTCGTCGTCTGCAG960                TAGCTTTGAAGTCATATTTGGCGATGGCTTCCATTCTGAGCGCTGCTCAGTGCTCAGCAG1020               CCTGAAGCAGGGGGAAGGGAGGGAGGAGGGCTGAGCCCCCGAATTCACTGGC1072                       (2) INFORMATION FOR SEQ ID NO:50:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 949 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                       GACTCTAGAGGATCCCCGGATCCGAGCTCGAATTCGGTTTTTATTATTGGCGTCAGCTAG60                 GACTATACGTGGCCTTAAACGTCATGCACTGATGGACAGAAGAGAAAAAAGGATGAAAAA120                AAAAGACAAAGGAGGGGAAAGAGGAGCAGCAGTGAAAATTTGTAATAAAAACTCTTCTTA180                ATTTATAGGTAAGTTTTGGCATTTTTAAATCCAACGCCCCCTCCCACCCCCTAAAGTTCC240                AACCAAAGTGAGAGGGTCACAGGGTGACCCGGCCAGGTGTTCTGCACTCCCTCACAGGCT300                GCTGTCAGAGGCAGCTTGTGGGTTTAATTCTTTTGTATGTGTTTTACATTTTTCACTTTC360                TTTAAATAATTGCTTCTTGACTCTTAGACGTTCCGGTTCACNGGGGGAGACATAATTCGC420                GGGGAAACATGCCGGTCTGCCCGTGGCAAGCTCCTTTCCACCAGTTGGGGTCTGAGTTAT480                CCATGACATGGATAAAATCTCCCCGGCGGAAGCCCAGCTCTCCATCCTCCTGGGGATCAA540                AGTCAAAGAGGGCCTGGACGTATGTCGGCTGCTGTGGCACCTGTTCTATGTCCCGCAGGA600                ATATCTGCTGGTTTCTGGAGACAGATGTAGATCTGTGATAATCCACCAGCTCATTCAAAG660                AATTGAACTTCACCACCAGAGGAAGTACTTCCCGGCTCCATCTCGGGAGCACCTTGAAAG720                TGCTGCACATCGTTCCAAACTTGACAGAGAGGGAGAAGTCCCCAGGAGCGCTCTCACTCT780                CTCGGATAAGAAAGGCCCCATCGTGCCGCTGTTTGCTAAGCATTTCTTCTGCCTTGGCTC840                TGGGGATTTTGCCAAAAAACCACGGATGTGGTTTCATTTCTATGTAGTTCTTGGGAATGA900                AGCCGTCTTTTCCATTAAGCTCTGCCAATTCGGGCCCGAATTCACTGGC949                           __________________________________________________________________________ 

What is claimed is:
 1. A recombinant nucleic acid encoding a protein containing the amino acid sequence shown in (SEQ ID NO:6).
 2. A recombinant nucleic acid according to claim 1, wherein the nucleic acid comprises a plasmid.
 3. A host transformed with the nucleic acid of claim
 1. 4. A recombinant nucleic acid which hybridizes under stringent conditions to the complement of the recombinant nucleic acid of claim
 1. 5. A recombinant nucleic acid according to claim 4, wherein the nucleic acid comprises a plasmid.
 6. A host containing the nucleic acid of claim
 4. 7. A recombinant nucleic acid which encodes the Src homology 2 peptide domain contained within the protein depicted in (SEQ ID NO:6).
 8. A host containing the nucleic acid of claim
 7. 9. A recombinant nucleic acid which encodes a peptide containing amino acid residues 60 to 158 depicted in (SEQ ID NO:6).
 10. A host containing the nucleic acid of claim
 9. 11. A recombinant nucleic acid which encodes a Src homology 3 peptide domain contained within the protein depicted in (SEQ ID NO:6).
 12. A host containing the nucleic acid of claim
 11. 13. A recombinant nucleic acid which encodes a peptide containing amino acid residues 5 to 54 depicted in (SEQ ID NO:6).
 14. A host containing the nucleic acid of claim
 13. 15. A recombinant nucleic acid which encodes a peptide containing amino acid residues 163 to 208 depicted in (SEQ ID NO:6).
 16. A host containing the nucleic acid of claim
 15. 17. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:6 and lacking the Src homology 2 peptide domain contained within the amino acid sequence depicted in SEQ ID NO:6.
 18. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:6 except for lacking a Src homology 3 peptide domain contained therein.
 19. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:6 and lacking at least one, but not more than two, of the following domains: the SH2 domain, the amino-terminal SH3 domain or the carboxy-terminal SH3 domain.
 20. A nucleic acid molecule encoding a fusion protein comprising the polypeptide of claim 19 fused to a heterologous polypeptide.
 21. An isolated nucleic acid molecule which encodes a peptide containing the following amino acid residues shown in SEQ ID NO:6: 1-161, 2-59, 2-217, 50-161, 50-217 or 156-217.
 22. A nucleic acid molecule which encoding a fusion protein comprising the peptide of claim 21 fused to a heterologous polypeptide.
 23. A process for preparing a protein containing an amino acid sequence shown in SEQ ID NO:6, comprising:(a) culturing a recombinant host according to claim 3, 4, 8, 10, 12, 14 or 16, such that said protein is expressed in recoverable amounts; and recovering said protein form said culture or host.
 24. A recombinant vector containing the nucleic acid molecule of claim 1, 4, 7, 9, 11, 13, 15, 17, 18, 19, 21, 20 or
 22. 25. An expression vector containing the nucleic acid molecule of claim 1, 4, 7, 9, 11, 13, 15, 17, 18, 19, 21, 20 or 22, operatively associated with regulatory sequence that controls expression of the nucleic acid in a host cell.
 26. A genetically engineered host cell containing the nucleic acid molecule of claim 1, 4, 7, 9, 11, 13, 15, 17, 18, 19, 21, 20 or
 22. 27. A genetically engineered host cell containing the nucleic acid molecule of claim 1, 4, 7, 9, 11, 13, 15, 17, 18, 19, 21, 20 or 22 operatively associated with a regulatory sequence that controls expression of the nucleic acid in a host cell.
 28. A process for preparing a protein containing an amino acid sequence shown in SEQ ID NO:6, comprising:(a) culturing a genetically engineered host cell according to claim 27, such that said protein is expressed in recoverable amounts; and (b) recovering said protein from said culture or host. 